CD127 binding proteins

ABSTRACT

Antigen binding proteins which bind to human IL-7 receptor (CD127) are provided. The antigen binding proteins are typically antibodies, and are useful in the treatment of diseases or disorders in humans, particularly autoimmune diseases such as multiple sclerosis.

This application claims the benefit of U.S. Provisional Application No.61/299,010 filed 28 Jan. 2010, which is incorporated herein in itsentirety.

FIELD OF THE INVENTION

The present invention relates to antigen binding proteins, in particularimmunoglobulins that specifically bind to the α-chain of the human IL-7receptor (CD127). The invention also concerns methods of treatingdiseases or disorders with said proteins, pharmaceutical compositionscomprising said proteins and methods of their manufacture. Other aspectsof the present invention will be apparent from the description below.

BACKGROUND OF THE INVENTION

Multiple Sclerosis (MS) is a chronic inflammatory, demyelinating diseasethat affects the central nervous system. In MS, it is believed thatinfiltrating inflammatory immune cells are involved in the destructionof oligodendrocytes, which are the cells responsible for creating andmaintaining a fatty layer, known as the myelin sheath. MS results in thethinning or complete loss of myelin. When the myelin is lost, theneurons can no longer effectively conduct their electrical signalsleading to numerous neurologic dysfunctions. Individuals with MS produceautoreactive T cells that participate in the formation of inflammatorylesions along the myelin sheath of nerve fibres. The cerebrospinal fluidof patients with active MS contains activated T cells, which infiltratethe brain tissue and cause characteristic inflammatory lesions,destroying the myelin. While the multiple sclerosis symptoms and courseof illness can vary from person to person, there are three forms of thedisease—relapsing-remitting MS, secondary progressive MS, and primaryprogressive MS.

In the early stages of MS, inflammatory attacks occur over shortintervals of acutely heightened disease activity. These episodes arefollowed by periods of recovery and remission. During the remissionperiod, the local swelling in the nervous system lesion resolves, theimmune cells become less active or inactive, and the myelin-producingcells remyelinate the axons. Nerve signalling improves, and thedisability caused by the inflammation becomes less severe or goes awayentirely. This phase of the disease is called relapsing-remitting MS(RRMS). The lesions do not all heal completely, though. Some remain as“chronic” lesions, which usually have a demyelinated core region whichlacks immune cells. Over time, the cells in the centre of such lesionsmostly die, although inflammation often continues at their edges. Thebrain can adapt well to the loss of some neurons, and permanentdisability may not occur for many years. However, more than 50% ofpatients with MS eventually enter a stage of progressive deterioration,called secondary progressive MS (SPMS). In this stage, the disease nolonger responds well to disease-modifying drugs, and patients'disabilities steadily worsen. The destruction of neurons from early inthe natural course of MS suggests that the progressive disabilities ofSPMS might be the result of an accumulated neuronal loss that eventuallyoverwhelms the brain's compensatory abilities. Primary progressive MS isa type of multiple sclerosis where there are no relapses, but over aperiod of years, there is gradual loss of physical and cognitivefunctions.

The goal of treatment in patients with relapsing-remitting multiplesclerosis is to reduce the frequency and severity of relapses (andthereby prevent exacerbations) as well as to prevent or postpone theonset of the progressive phase of the disease. To achieve this goal, inthe past especially, immunomodulatory or immunosuppressive drugs havebeen used, but they have never found widespread acceptance owing tolimited efficacy and considerable toxicity. For example, largerandomized controlled trials have been performed successfully withinterferon beta-1a, interferon beta-1b, and glatiramer acetate.

Both altered autoimmune T cell responses and dysfunction of theregulatory network of the immune system play an important role in humanautoimmune pathologies, such as MS and rheumatoid arthritis (Kuchroo etal., (2002) Annu. Rev. Immunol. 20:101-123; Sospedra and Martin (2005)Annu. Rev. Immunol. 23: 683-747; Toh and Miossec (2007) Curr. Opin.Rheumatol. 19:284-288).

Although the aetiology and pathogenesis of MS remain unknown, it isgenerally considered an autoimmune pathology in which autoreactive Tcells of pathogenic potential, such as T_(H)1 and T_(H)17 cells, arethought to play an important role. There is evidence that these effectorT cells are activated in vivo during the disease process and areattributable to the central nervous system (CNS) inflammation. There isalso evidence that these T cells mediate destruction ofmyelin-expressing cells in lesions of EAE and MS during the active phaseof the disease. On the other hand, regulatory T cells (T_(reg)) thatnormally keep pathogenic T_(H)1 and T_(H)17 cells in check are deficientin patients with MS, further tilting the immune system toward anpro-inflammatory state.

Three separate groups recently reported the results of genome widesingle nucleotide polymorphisms (SNPs) scanning in a total of 17,947donors with or without MS. After scanning 334,923 SNPs, they found ahighly significant association (overall P=2.9×10⁻⁷) of a nonsynonymouscoding SNP in the human IL-7 receptor alpha chain (IL-7Rα) with MSsusceptibility. The SNP corresponds to a change from T to C in exon 6 ofCD127 (also known as IL-7Rα). This change enhances the chance of exon 6skipping during RNA splicing, resulting in a soluble form of CD127.Furthermore, expressions of CD127 and IL-7 RNAs in the cerebrospinalfluids (CSFs) of MS patients are significantly higher relative to CSFsof patients with other neurological disorders.

IL-7 and IL-7 receptor (IL-7R) are known to play an important role in Tcell and B cell development and homeostasis mainly in a thymicenvironment. Indeed, thymic stromal cells, fetal thymus, and bone marroware sites of IL-7 of production. The IL-7 receptor consists of twosubunits, CD127 and a common chain (gamma chain or γc) which is sharedby receptors of IL-2, IL-4, IL-9, IL-15, and IL-21.

CD127 is also known as IL-7 receptor alpha (IL-7Rα) and p90 IL-7R. HumanCD127 (Swiss Prot accession number P16871) has a total of 459 aminoacids (20 signal sequence). It comprises a 219 amino acid extra cellularregion, a 25 amino acid transmembrane region and a 195 amino acidintracellular region. The numbering of residues within CD127, as usedherein (e.g. for the description of antibody epitopes) is based on thefull length protein, including signal sequence residues. CD127 may existin four isoforms, the isoform H20

(Swissprot accession number P16871-1) has the following amino acidsequence (including signal sequence):

(SEQ ID NO: 1) MTILGTTFGM VFSLLQVVSG ESGYAQNGDL EDAELDDYSFSCYSQLEVNG SQHSLTCAFE DPDVNTTNLE FEICGALVEVKCLNFRKLQE IYFIETKKFL LIGKSNICVK VGEKSLTCKKIDLTTIVKPE APFDLSVIYR EGANDFVVTF NTSHLQKKYVKVLMHDVAYR QEKDENKWTH VNLSSTKLTL LQRKLQPAAMYEIKVRSIPD HYFKGFWSEW SPSYYFRTPE INNSSGEMDPILLTISILSF FSVALLVILA CVLWKKRIKP IVWPSLPDHKKTLEHLCKKP RKNLNVSFNP ESFLDCQIHR VDDIQARDEVEGFLQDTFPQ QLEESEKQRL GGDVQSPNCP SEDVVVTPESFGRDSSLTCL AGNVSACDAP ILSSSRSLDC RESGKNGPHVYQDLLLSLGT TNSTLPPPFS LQSGILTLNP VAQGQPILTS LGSNQEEAYV TMSSFYQNQ

CD127 is also found in the receptor of thymic stromal derivedlymphopoietin (TSLP). The TSLP receptor is a heterodimer of CD127 andcytokine receptor-like factor 2 (CRLF2).

Binding of IL-7 to the IL-7R activates multiple signalling pathwaysincluding the activation of JAK kinases 1 and 3 leading to thephosphorylation and activation of Stat5. This pathway is crucial to thesurvival of thymic developing T cell precursors because Stat5 activationis required in the induction of the anti-apoptotic protein Bcl-2 and theprevention of the pro-apoptotic protein Bax entry into themitochondrion. Another IL-7R mediated pathway is the activation of P13kinase, resulting in the phosphorylation of the pro-apoptotic proteinBad and its cytoplasm retention. CD127 is expressed in peripheralresting and memory T cells. The mechanism of IL-7 regulation of T cellsurvival and homeostasis and the source of IL-7 in the periphery are notcompletely understood. Furthermore, its potential role in thedifferentiation and function of pathogenic T cells in autoimmune diseaseis poorly studied and largely unknown. There are few reports suggestingthat IL-7 may contribute to the pathogenesis of autoimmune diseases.

Recently, Liu and colleagues (Liu et al, (2010) Nature Medicine16:191-197) have described the role of IL-7 in T_(H)17 survival andexpansion. Murine anti-CD127 antibodies (including the anti-CD127antibodies 1A11 and 6A3) and their role in the treatment of MS and otherautoimmune diseases have been described in PCT application numberPCT/US2009/053136.

It is desirable to isolate and develop further monoclonal antibodiesthat bind to and/or inhibit the biological effect of human CD127. Suchantibodies are likely to be therapeutically useful in the treatment ofMS and other inflammatory and autoimmune diseases and disorders,particularly those in which pathogenic T_(H)17 cells have beenimplicated.

SUMMARY OF THE INVENTION

The invention provides antigen binding proteins which specifically bindto CD127. The antigen binding proteins can be used in therapeuticmethods, in particular, in the treatment or prevention of diseases inwhich pathogenic T_(H)17 cells are implicated. The antigen bindingproteins may bind to CD127 and inhibit, e.g. neutralize, the biologicalfunction of CD127.

In a first aspect, the invention provides an antigen binding proteincomprising a heavy chain variable domain having an amino acid sequenceas set out in SEQ ID NO:13, and a light chain variable domain having anamino acid sequence as set out in SEQ ID NO:22.

The antigen binding protein may comprise a heavy chain having an aminoacid sequence as set out in SEQ ID NO:114 or SEQ ID NO:118. The antigenbinding protein may further comprise a light chain having an amino acidsequence as set out in SEQ ID NO:115.

The invention also provides a nucleic acid molecule encoding an antibodyor antigen binding protein according to the invention, an expressionvector comprising such a nucleic acid molecule, and a recombinant hostcell comprising such an expression vector.

In a further aspect, the invention provides an antibody or antigenbinding protein expressed by a host cell.

The invention also provides a method for the production of an antibodyor antigen binding protein according to claim 1, comprising the step ofculturing a host cell according to the invention in a medium to producethe antigen binding protein, and isolating or purifying the antigenbinding protein therefrom. The culturing step may be performed inconditions conducive for expression of the antibody or antigen bindingprotein from the host cell, and secretion of the antibody from the cell.

In a further aspect, the invention provides a pharmaceutical compositioncomprising an antigen binding protein according to the invention and apharmaceutically acceptable carrier or excipient.

In a still further aspect, the invention provides a method of treating asubject having an autoimmune or inflammatory disease, comprising thestep of administering to the subject an antigen binding proteinaccording to the invention. In an embodiment, the autoimmune orinflammatory disease is multiple sclerosis.

The antigen binding protein may be an antibody, in particular, ahumanised or human antibody, or an antigen binding fragment thereof. Theantigen binding protein of the invention may not inhibit TSLPsignalling. In an embodiment, the antigen binding protein does notinhibit TSLP signalling.

The invention also provides a nucleic acid molecule encoding an antigenbinding protein of the present invention. In an embodiment, theinvention provides nucleic acid molecules of SEQ ID NO:32, SEQ IDNO:108, and SEQ ID NO:119. The invention also provides an expressionvector comprising a nucleic acid molecule as defined herein, and arecombinant host cell comprising an expression vector as defined herein.The expression vector may comprise the nucleic acid molecule of SEQ IDNO:32, SEQ ID NO:108, and SEQ ID NO:119. In an embodiment, theexpression vector comprises a nucleic acid molecule which encodes anantigen binding protein as hereinbefore described. In anotherembodiment, the invention provides a host cell comprising an expressionvector as hereinbefore described. In a further embodiment, the inventionprovides an antibody expressed by host cell as hereinbefore described.

The invention also provides a method for the production of an antigenbinding protein of the present invention which method comprises the stepof culturing a host cell as defined above and recovering the antigenbinding protein.

The invention also provides an antibody or antigen binding proteinaccording to the invention which is expressed by a host cell comprisinga nucleic acid sequence or sequence encoding an antibody or antigenbinding fragment according to the invention.

The invention also provides a pharmaceutical composition comprising anantigen binding protein of the present invention and a pharmaceuticallyacceptable carrier or excipient.

The invention also provides a method of treating a subject afflictedwith an autoimmune or inflammatory disease, which method comprises thestep of administering to the subject an antigen binding protein of thepresent invention.

The invention also provides a method of treating a subject afflictedwith a disease in which pathogenic T_(H)17 cells are implicated, whichmethod comprises the step of administering to the subject an antigenbinding protein of the present invention.

The invention also provides a method of treating a subject afflictedwith a disease associated with upregulated expression of IL-17, whichmethod comprises the step of administering to the subject an antigenbinding protein of the present invention.

In particular, the autoimmune or inflammatory disease, the disease inwhich pathogenic T_(H)17 cells are implicated, or the disease associatedwith up-regulated expression of IL17 may be multiple sclerosis (MS),SLE, rheumatoid arthritis, Behcet's disease or asthma. In an embodiment,the antigen binding protein of the invention will be useful in a methodof treating multiple sclerosis. Other diseases which may be treated bythe administration of the antigen binding proteins of the invention aredescribed herein.

The invention also provides an antigen binding protein as describedherein for use in the treatment of a subject afflicted with anautoimmune or inflammatory disease; a disease in which pathogenicT_(H)17 cells are implicated; or a disease associated with up-regulatedexpression of IL17.

The invention provides the use of an antigen binding protein asdescribed herein in the manufacture of a medicament for use in thetreatment of, a subject afflicted with an autoimmune or inflammatorydisease; a disease in which pathogenic T_(H)17 cells are implicated; ora disease associated with up-regulated expression of IL17.

Other aspects and embodiments of the invention will be apparent from thedetailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the complement dependent cytotoxicity of the anti-IL7R mAb1A11 H3L4 on HEK293 cells expressing hIL-7R.

FIG. 2 shows the antibody dependent cell-mediated cytotoxicity of thehumanised anti-IL7R mAb 1A11 H3L4 and the Fc-disabled anti-IL7R mAb(1A11 H3L4Fc) on HEK293 cells expressing hIL-7R, in the presence ofperipheral blood mononuclear cells.

FIGS. 3A and 3B show the inhibition of IL-7-induced STAT5phosphorylation by 1A11 H3L4 in human PBMC provided by two separatedonors.

FIGS. 4A to 4D show the inhibition of IL-7-induced IL-17 production indifferentiated human Th17 cells by 1A11 H3L4 (in four different donors).

FIGS. 5A to 5E show that 1A11 H3L4 does not affect TSLP-induction ofTARC (thymus and activation-regulated chemokine).

DETAILED DESCRIPTION OF THE INVENTION

IL-7/IL-7R signalling is critically required for survival and expansionof committed T_(H)17 cells in both mouse and human systems, while itsrole in T_(H)17 differentiation is not essential compared to that ofIL-6 (Liu et al, (2010) Nature Medicine 16:191-197). Surprisingly, thein vivo effect on the immune system by IL-7R antagonism is highlyselective in EAE, an animal model for multiple sclerosis, affectingT_(H)17 cells and, to a lesser extent, T_(H)1 cells predominantly of thememory phenotype, and sparing T_(reg) cells. This selectivity appears toplay an important role in rebalancing the ratio of pathogenic T_(H)17cells and T_(reg) cells by IL-7R antagonism in EAE and is attributableto the treatment efficacy.

The role of IL-7/IL-7R signalling in T_(H)17 cell survival and expansionsupports the treatment efficacy of IL-7R antagonism in human autoimmunediseases, such as MS. IL-7 neutralization or IL-7R antagonism is likelyto have unique therapeutic advantages. On one hand, the treatment offersthe selectivity that distinguishes pathogenic T_(H)1 and T_(H)17 cellsfrom T_(reg) and unrelated immune cells. On the other hand, additionaltherapeutic advantages of IL-7R antagonism involve its selective effecton survival and expansion of differentiated T_(H)17 as opposed toT_(H)17 differentiation. It is conceivable that targeting in vivomaintenance of committed T_(H)17 versus T_(H)17 differentiation is moreefficacious in a therapeutic context. Inhibition of IL-7 receptormediated signalling therefore provides a promising therapeuticintervention for the treatment of autoimmune or inflammatory diseases.

The term IL-7R mediated signalling, as used herein, means the biologicaleffect instigated by the IL-7 receptor complex when bound by its ligand,IL-7. IL-7R mediated signalling therefore includes, but is notnecessarily limited to, one or more, or all, of IL-7 inducedphosphorylation of STAT-5, IL-7 induced expansion of T_(H)17 cells andIL-7 induced survival of T_(H)17 cells.

Murine antibodies 1A11 and 6A3 are described in patent applicationnumber PCT/US2009/053136 (WO2010/017468). These antibodies specificallybind to the alpha chain of the human IL-7 receptor, CD127 (SEQ ID NO:1).The variable domains of these antibodies are described in SEQ ID NO:8and 9 (V_(H), Vκ 1A11, respectively) and SEQ ID NO:46 and 47 (V_(H), Vκ6A3, respectively).

The present invention provides antigen binding proteins comprising oneor more of the complementarity determining regions (CDRs) of 1A11 or6A3, and variants thereof. The antigen binding proteins may bind to andneutralise IL-7R signalling. In one embodiment, the invention provideshumanised antibodies, comprising the from one to six of the CDRs frommurine antibodies 1A11 or 6A3 (the donor antibody) in an a humanacceptor antibody.

The term “antigen binding protein” as used herein refers to antibodies,antibody fragments and other protein constructs, such as domains, whichare capable of binding to CD127. In an embodiment, the antigen bindingprotein is an antibody.

The term “antibody” is used herein in the broadest sense to refer tomolecules with an immunoglobulin-like domain and includes monoclonal,recombinant, polyclonal, chimeric, humanised, bispecific andheteroconjugate antibodies; a single variable domain, a domain antibody,antigen binding fragments, immunologically effective fragments, singlechain Fv, diabodies, Tandabs™, etc (for a summary of alternative“antibody” formats see Holliger and Hudson, Nature Biotechnology, 2005,Vol 23, No. 9, 1126-1136).

The phrase “single variable domain” refers to an antigen binding proteinvariable domain (for example, V_(H), V_(HH), V_(L)) that specificallybinds an antigen or epitope independently of a different variable regionor domain.

A “domain antibody” or “dAb” may be considered the same as a “singlevariable domain” which is capable of binding to an antigen. A singlevariable domain may be a human antibody variable domain, but alsoincludes single antibody variable domains from other species such asrodent (for example, as disclosed in WO 00/29004), nurse shark andCamelid V_(HH) dAbs. Camelid V_(HH) are immunoglobulin single variabledomain polypeptides that are derived from species including camel,llama, alpaca, dromedary, and guanaco, which produce heavy chainantibodies naturally devoid of light chains. Such V_(HH) domains may behumanised according to standard techniques available in the art, andsuch domains are considered to be “domain antibodies”. As used hereinV_(H) includes camelid V_(HH) domains.

As used herein the term “domain” refers to a folded protein structurewhich has tertiary structure independent of the rest of the protein.Generally, domains are responsible for discrete functional properties ofproteins, and in many cases may be added, removed or transferred toother proteins without loss of function of the remainder of the proteinand/or of the domain. A “single variable domain” is a folded polypeptidedomain comprising sequences characteristic of antibody variable domains.It therefore includes complete antibody variable domains and modifiedvariable domains, for example, in which one or more loops have beenreplaced by sequences which are not characteristic of antibody variabledomains, or antibody variable domains which have been truncated orcomprise N- or C-terminal extensions, as well as folded fragments ofvariable domains which retain at least the binding activity andspecificity of the full-length domain. A domain can bind an antigen orepitope independently of a different variable region or domain.

An antigen binding fragment may be provided by means of arrangement ofone or more CDRs on non-antibody protein scaffolds such as a domain. Anon-antibody protein scaffold or domain is one that has been subjectedto protein engineering in order to obtain binding to a ligand other thanits natural ligand, for example a domain which is a derivative of ascaffold selected from: CTLA-4 (Evibody); lipocalin; Protein A derivedmolecules such as Z-domain of Protein A (Affibody, SpA), A-domain(Avimer/Maxibody); heat shock proteins such as GroEI and GroES;transferrin (trans-body); ankyrin repeat protein (DARPin); peptideaptamer; C-type lectin domain (Tetranectin); human γ-crystallin andhuman ubiquitin (affilins); PDZ domains; scorpion toxinkunitz typedomains of human protease inhibitors; and fibronectin (adnectin); whichhas been subjected to protein engineering in order to obtain binding toa ligand other than its natural ligand.

CTLA-4 (Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-familyreceptor expressed on mainly CD4+ T-cells. Its extracellular domain hasa variable domain-like Ig fold. Loops corresponding to CDRs ofantibodies can be substituted with heterologous sequence to conferdifferent binding properties. CTLA-4 molecules engineered to havedifferent binding specificities are also known as Evibodies. For furtherdetails see Journal of Immunological Methods 248 (1-2), 31-45 (2001).

Lipocalins are a family of extracellular proteins which transport smallhydrophobic molecules such as steroids, bilins, retinoids and lipids.They have a rigid β-sheet secondary structure with a number of loops atthe open end of the canonical structure which can be engineered to bindto different target antigens. Anticalins are between 160-180 amino acidsin size, and are derived from lipocalins. For further details seeBiochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No. 7,250,297B1 andUS20070224633.

An affibody is a scaffold derived from Protein A of Staphylococcusaureus which can be engineered to bind to an antigen. The domainconsists of a three-helical bundle of approximately 58 amino acids.Libraries have been generated by randomisation of surface residues. Forfurther details see Protein Eng. Des. Sel. 17, 455-462 (2004) andEP1641818A1.

Avimers are multidomain proteins derived from the A-domain scaffoldfamily. The native domains of approximately 35 amino acids adopt adefined disulphide bonded structure. Diversity is generated by shufflingof the natural variation exhibited by the family of A-domains. Forfurther details see Nature Biotechnology 23(12), 1556-1561 (2005) andExpert Opinion on Investigational Drugs 16(6), 909-917 (June 2007).

A transferrin is a monomeric serum transport glycoprotein. Transferrinscan be engineered to bind different target antigens by insertion ofpeptide sequences, such as one or more CDRs, in a permissive surfaceloop. Examples of engineered transferrin scaffolds include theTrans-body. For further details see J. Biol. Chem. 274, 24066-24073(1999).

Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrinwhich is a family of proteins that mediate attachment of integralmembrane proteins to the cytoskeleton. A single ankyrin repeat is a 33residue motif consisting of two α-helices and a β-turn. They can beengineered to bind different target antigens by: randomising residues inthe first α-helix and a β-turn of each repeat; or insertion of peptidesequences, such as one or more CDRs. Their binding interface can beincreased by increasing the number of modules (a method of affinitymaturation). For further details see J. Mol. Biol. 332, 489-503 (2003),PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007)and US20040132028A1.

Fibronectin is a scaffold which can be engineered to bind to antigen.Adnectins consists of a backbone of the natural amino acid sequence ofthe 10th domain of the 15 repeating units of human fibronectin type III(FN3). Three loops at one end of the β-sandwich can be engineered toenable an Adnectin to specifically recognize a therapeutic target ofinterest. For further details see Protein Eng. Des. Sel. 18, 435-444(2005), US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1.

Peptide aptamers are combinatorial recognition molecules that consist ofa constant scaffold protein, typically thioredoxin (TrxA) which containsa constrained variable peptide loop inserted at the active site. Forfurther details see Expert Opin. Biol. Ther. 5, 783-797 (2005).

Microbodies are derived from naturally occurring microproteins of 25-50amino acids in length which contain 3-4 cysteine bridges; examples ofmicroproteins include KalataB1 and conotoxin and knottins. Themicroproteins have a loop which can be engineered to include up to 25amino acids without affecting the overall fold of the microprotein. Forfurther details of engineered knottin domains, see WO2008098796.

Other binding domains include proteins which have been used as ascaffold to engineer different target antigen binding properties includehuman γ-crystallin and human ubiquitin (affilins), kunitz type domainsof human protease inhibitors, PDZ-domains of the Ras-binding proteinAF-6, scorpion toxins (charybdotoxin), C-type lectin domain(tetranectins) are reviewed in Chapter 7—Non-Antibody Scaffolds fromHandbook of Therapeutic Antibodies (2007, edited by Stefan Dubel) andProtein Science 15:14-27 (2006). Binding domains of the presentinvention could be derived from any of these alternative protein domainsand any combination of the CDRs of the present invention grafted ontothe domain.

An antigen binding fragment or an immunologically effective fragment maycomprise partial heavy or light chain variable sequences. Fragments areat least 5, 6, 8 or 10 amino acids in length. Alternatively thefragments are at least 15, at least 20, at least 50, at least 75, or atleast 100 amino acids in length.

The term “specifically binds” as used throughout the presentspecification in relation to antigen binding proteins means that theantigen binding protein binds to CD127 with no or insignificant bindingto other (for example, unrelated) proteins. The term however does notexclude the fact that the antigen binding proteins may also becross-reactive with CD127 from other species, such as murine CD127,cynomolgus monkey (Macaca fascicularis) or marmoset CD127. In anembodiment, the antigen binding protein binds to both cynomolgus monkeyand marmoset CD127. The antigen binding proteins described herein maybind to human CD127 with at least 2, 5, 10, 50, 100, or 1000 foldgreater affinity than they bind to CD127 from other species.

The binding affinity or equilibrium dissociation constant (K_(D)) of theantigen binding protein-CD127 interaction may be 100 nM or less, 10 nMor less, 2 nM or less or 1 nM or less. Alternatively the K_(D) may bebetween 5 and 10 nM; or between 1 and 2 nM. The K_(D) may be between 1pM and 500 pM; or between 500 pM and 1 nM. The binding affinity of theantigen binding protein is determined by the association rate constant(k_(a)) and the dissociation rate constant (k_(d)) (K_(D)=k_(d)/k_(a)).The binding affinity may be measured by BIACORE™, for example by antigencapture with CD127 coupled onto a CM5 chip by primary amine coupling andantibody capture onto this surface. The BIACORE™ method described inExample 4 may be used to measure binding affinity. Alternatively, thebinding affinity can be measured by FORTEbio, for example by antigencapture with CD127 coupled onto a CM5 needle by primary amine couplingand antibody capture onto this surface.

The k_(d) may be 1×10⁻³ s⁻¹ or less, 1×10⁻⁴ s⁻¹ or less, or 1×10⁻⁵ s⁻¹or less. The k_(d) may be between 1×10⁻⁵ s⁻¹ and 1×10⁻⁴ s⁻¹; or between1×10⁻⁴ s⁻¹ and 1×10⁻³ s⁻¹. A slow k_(d) may result in a slowdissociation of the antigen binding protein-ligand complex and improvedneutralisation of the ligand.

It will be apparent to those skilled in the art that the term “derived”is intended to define not only the source in the sense of it being thephysical origin for the material but also to define material which isstructurally identical to the material but which does not originate fromthe reference source. Thus “residues found in the donor antibody” neednot necessarily have been purified from the donor antibody.

By isolated it is intended that the molecule, such as an antigen bindingprotein, is removed from the environment in which it may be found innature. For example, the molecule may be purified away from substanceswith which it would normally exist in nature. For example, the antigenbinding protein can be purified to at least 95%, 96%, 97%, 98% or 99%,or greater with respect to a culture media containing the antigenbinding protein.

A “chimeric antibody” refers to a type of engineered antibody whichcontains a naturally-occurring variable region (light chain and heavychains) derived from a donor antibody in association with light andheavy chain constant regions derived from an acceptor antibody.

A “humanised antibody” refers to a type of engineered antibody havingone or more of its CDRs derived from a non-human donor immunoglobulin,the remaining immunoglobulin-derived parts of the molecule being derivedfrom one or more human immunoglobulin(s). In addition, framework supportresidues may be altered to preserve binding affinity (see, e.g., Queenet al. Proc. Natl. Acad Sci USA, 86:10029-10032 (1989), Hodgson et al.Bio/Technology, 9:421 (1991)). A suitable human acceptor antibody may beone selected from a conventional database, e.g., the KABAT® database,Los Alamos database, and Swiss Protein database, by homology to thenucleotide and amino acid sequences of the donor antibody. A humanantibody characterized by a homology to the framework regions of thedonor antibody (on an amino acid basis) may be suitable to provide aheavy chain constant region and/or a heavy chain variable frameworkregion for insertion of the donor CDRs. A suitable acceptor antibodycapable of donating light chain constant or variable framework regionsmay be selected in a similar manner. It should be noted that theacceptor antibody heavy and light chains are not required to originatefrom the same acceptor antibody. The prior art describes several ways ofproducing such humanised antibodies, see for example EP-A-0239400 andEP-A-054951.

The term “donor antibody” refers to an antibody which contributes theamino acid sequences of its variable regions, one or more CDRs, or otherfunctional fragments or analogs thereof to a first immunoglobulinpartner. The donor therefore provides the altered immunoglobulin codingregion and resulting expressed altered antibody with the antigenicspecificity and neutralising activity characteristic of the donorantibody.

The term “acceptor antibody” refers to an antibody which is heterologousto the donor antibody, which contributes all (or any portion) of theamino acid sequences encoding its heavy and/or light chain frameworkregions and/or its heavy and/or light chain constant regions to thefirst immunoglobulin partner. A human antibody may be the acceptorantibody.

The terms “V_(H)” and “V_(L)” are used herein to refer to the heavychain variable region and light chain variable region respectively of anantigen binding protein. Vκ is also used to refer to the variable lightchain domain.

“CDRs” are defined as the complementarity determining region amino acidsequences of an antigen binding protein. These are the hypervariableregions of immunoglobulin heavy and light chains. There are three heavychain and three light chain CDRs (or CDR regions) in the variableportion of an immunoglobulin. Thus, “CDRs” as used herein refers to allthree heavy chain CDRs, all three light chain CDRs, all heavy and lightchain CDRs, or at least two CDRs.

Throughout this specification, amino acid residues in variable domainsequences and full length antibody sequences are numbered according tothe Kabat numbering convention, unless otherwise specified. Similarly,the terms “CDR”, “CDRL1”, “CDRL2”, “CDRL3”, “CDRH1”, “CDRH2”, “CDRH3”used in the Examples follow the Kabat numbering convention. For furtherinformation, see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 4th Ed., U.S. Department of Health and Human Services,National Institutes of Health (1987).

It will be apparent to those skilled in the art that there arealternative numbering conventions for amino acid residues in variabledomain sequences and full length antibody sequences. There are alsoalternative numbering conventions for CDR sequences, for example thoseset out in Chothia et al. (1989) Nature 342: 877-883. The structure andprotein folding of the antibody may mean that other residues areconsidered part of the CDR sequence and would be understood to be so bya skilled person. Therefore, the term “corresponding CDR” is used hereinto refer to a CDR sequence using any numbering convention, for examplethose set out in Table 1.

Other numbering conventions for CDR sequences available to a skilledperson include “AbM” (University of Bath) and “contact” (UniversityCollege London) methods. The minimum overlapping region using at leasttwo of the Kabat, Chothia, AbM and contact methods can be determined toprovide the “minimum binding unit”. The minimum binding unit may be asub-portion of a CDR.

Table 1 below represents one definition using each numbering conventionfor each CDR or binding unit. The Kabat numbering scheme is used inTable 1 to number the variable domain amino acid sequence. It should benoted that some of the CDR definitions may vary depending on theindividual publication used.

TABLE 1 Minimum Kabat Chothia AbM Contact binding CDR CDR CDR CDR unitH1 31-35/ 26-32/ 26-35/ 30-35/ 31-32 35A/35B 33/34 35A/35B 35A/35B H250-65 52-56 50-58 47-58 52-56 H3 95-102 95-102 95-102 93-101 95-101 L124-34 24-34 24-34 30-36 30-34 L2 50-56 50-56 50-56 46-55 50-55 L3 89-9789-97 89-97 89-96 89-96

As used herein, the term “antigen binding site” refers to a site on anantigen binding protein which is capable of specifically binding to anantigen. This may be a single domain (for example, an epitope-bindingdomain), or single-chain Fv (ScFv) domains or it may be pairedV_(H)/V_(L) domains as can be found on a standard antibody.

The term “epitope” as used herein refers to that portion of the antigenthat makes contact with a particular binding domain of the antigenbinding protein. An epitope may be linear, comprising an essentiallylinear amino acid sequence from the antigen. Alternatively, an epitopemay be conformational or discontinuous. For example, a conformationalepitope comprises amino acid residues which require an element ofstructural constraint. A discontinuous epitope comprises amino acidresidues that are separated by other sequences, i.e. not in a continuoussequence in the antigen's primary sequence. In the context of theantigen's tertiary and quaternary structure, the residues of adiscontinuous epitope are near enough to each other to be bound by anantigen binding protein.

For nucleotide and amino acid sequences, the term “identical” or“sequence identity” indicates the degree of identity between two nucleicacid or two amino acid sequences, and if required when optimally alignedand compared with appropriate insertions or deletions.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical positions/total number of positions times 100), taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm, as described below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package, using a NWSgapdna.CMPmatrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide oramino acid sequences can also be determined using the algorithm of E.Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which hasbeen incorporated into the ALIGN program (Version 2.0), using a PAM120weight residue table, a gap length penalty of 12 and a gap penalty of 4.In addition, the percent identity between two amino acid sequences canbe determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453(1970)) algorithm which has been incorporated into the GAP program inthe GCG software package, using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

In one method, a polynucleotide sequence may be identical to a referencepolynucleotide sequence as described herein (see for example SEQ ID NO:30-39, SEQ ID NO:76-105), that is be 100% identical, or it may includeup to a certain integer number of nucleotide alterations as compared tothe reference sequence, such as at least 50, 60, 70, 75, 80, 85, 90, 95,98, or 99% identical. Such alterations are selected from at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleotide alterations is determined by multiplying the total number ofnucleotides in the reference polynucleotide sequence as described herein(see for example SEQ ID NO: 30-39, SEQ ID NO:76-105), by the numericalpercent of the respective percent identity (divided by 100) andsubtracting that product from said total number of nucleotides in thereference polynucleotide sequence as described herein (see for exampleSEQ ID NO: 30-39, SEQ ID NO:76-105), or:n _(n) ≦x _(n)−(x _(n) ·y),wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in the reference polynucleotide sequence asdescribed herein (see for example SEQ ID NO: 30-39, SEQ ID NO:76-105),and y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.75 for 75%, 0.80for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.98 for 98%, 0.99for 99% or 1.00 for 100%, · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).

Similarly, a polypeptide sequence may be identical to a polypeptidereference sequence as described herein (see for example SEQ ID NO:1-29,SEQ ID NO:40-75) that is be 100% identical, or it may include up to acertain integer number of amino acid alterations as compared to thereference sequence such that the % identity is less than 100%, such asat least 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% identical. Suchalterations are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion, and wherein said alterations may occur atthe amino- or carboxy-terminal positions of the reference polypeptidesequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence. Thenumber of amino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in the polypeptide sequenceencoded by the polypeptide reference sequence as described herein (seefor example SEQ ID NO:1-29, SEQ ID NO:40-75) by the numerical percent ofthe respective percent identity (divided by 100) and then subtractingthat product from said total number of amino acids in the polypeptidereference sequence as described herein (see for example SEQ ID NO:1-29,SEQ ID NO:40-75), or:n _(a) ≦x _(a)−(x _(a) ·y),wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in the reference polypeptide sequence asdescribed herein (see for example SEQ ID NO:1-29, SEQ ID NO:40-75), andy is, 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.75 for 75%, 0.80 for80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.98 for 98%, 0.99 for99%, or 1.00 for 100%, · is the symbol for the multiplication operator,and wherein any non-integer product of x_(a) and y is rounded down tothe nearest integer prior to subtracting it from x_(a).

The % identity may be determined across the full length of the sequence,or any fragments thereof; and with or without any insertions ordeletions.

The terms “peptide”, “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues. A peptide may bemonomeric or polymeric.

It is well recognised in the art that certain amino acid substitutionsare regarded as being “conservative”. Amino acids are divided intogroups based on common side-chain properties and substitutions withingroups that maintain all or substantially all of the binding affinity ofthe antigen binding protein are regarded as conservative substitutions,see Table 2 below:

TABLE 2 Side chain Members Hydrophobic met, ala, val, leu, ile Neutralhydrophilic cys, ser, thr Acidic asp, glu Basic asn, gln, his, lys, argResidues that influence chain orientation gly, pro Aromatic trp, tyr,phe

It may be desirable to modify the effector function so of the antigenbinding fragment—for instance, to enhance ADCC or CDC, half life, etc.

In an embodiment, the antigen binding proteins of the invention may beFc disabled. One way to achieve Fc disablement comprises thesubstitutions of alanine residues at positions 235 and 237 (EU indexnumbering) of the heavy chain constant region. Alternatively, theantigen binding protein may be Fc enabled and not comprise the alaninesubstitutions at positions 235 and 237.

The antigen binding protein may have a half life of at least 6 hours, atleast 1 day, at least 2 days, at least 3 days, at least 4 days, at least5 days, at least 7 days, or at least 9 days in vivo in humans, or in amurine animal model.

The antigen binding protein may be derived from rat, mouse, primate(e.g. cynomolgus, Old World monkey or Great Ape) or human. The antigenbinding protein may be a human, humanised or chimeric antibody. Theantigen binding protein may comprise a constant region, which may be ofany isotype or subclass. The constant region may be of the IgG isotype,for example IgG1, IgG2, IgG3, IgG4 or variants thereof. The antigenbinding protein constant region may be IgG1.

Mutational changes to the Fc effector portion of the antibody can beused to change the affinity of the interaction between the FcRn andantibody to modulate antibody turnover. The half life of the antibodycan be extended in vivo. This could be beneficial to patient populationsas maximal dose amounts and maximal dosing frequencies could be achievedas a result of maintaining in vivo IC50 for longer periods of time. TheFc effector function of the antibody may be removed, in its entirety orin part, since it may not be desirable to kill those cells expressingCD127. This removal may result in an increased safety profile.

The antigen binding protein comprising a constant region may havereduced ADCC and/or complement activation or effector functionality. Theconstant domain may comprise a naturally disabled constant region ofIgG2 or IgG4 isotype or a mutated IgG1 constant domain. Examples ofsuitable modifications are described in EP0307434. One way to achieve Fcdisablement comprises the substitutions of alanine residues at positions235 and 237 (EU index numbering) of the heavy chain constant region.

The antigen binding protein may comprise one or more modificationsselected from a mutated constant domain such that the antibody hasenhanced effector functions/ADCC and/or complement activation. Examplesof suitable modifications are described in Shields et al. J. Biol. Chem.(2001) 276:6591-6604, Lazar et al. PNAS (2006) 103:4005-4010 and U.S.Pat. No. 6,737,056, WO2004063351 and WO2004029207.

The antigen binding protein may comprise a constant domain with analtered glycosylation profile such that the antigen binding protein hasenhanced effector functions/ADCC and/or complement activation. Examplesof suitable methodologies to produce an antigen binding protein with analtered glycosylation profile are described in WO2003/011878,WO2006/014679 and EP1229125.

The CD127 polypeptide to which the antigen binding protein binds may bea recombinant polypeptide, and may comprise the extracellular domain(ECD), optionally fused to another protein, such as an Fc domain, or maycomprise the full length CD127 protein. CD127 may be in solution or maybe attached to a solid surface. For example, CD127 may be attached tobeads such as magnetic beads. CD127 may be biotinylated. The biotinmolecule conjugated to CD127 may be used to immobilize CD127 on a solidsurface by coupling biotinstreptavidin on the solid surface.

The present invention also provides a nucleic acid molecule whichencodes an antigen binding protein as described herein. The nucleic acidmolecule may comprise a sequence encoding (i) one or more CDRHs, theheavy chain variable sequence, or the full length heavy chain sequence;and (ii) one or more CDRLs, the light chain variable sequence, or thefull length light chain sequence, with (i) and (ii) on the same nucleicacid molecule. Alternatively, the nucleic acid molecule which encodes anantigen binding protein described herein may comprise sequences encoding(a) one or more CDRHs, the heavy chain variable sequence, or the fulllength heavy chain sequence; or (b) one or more CDRLs, the light chainvariable sequence, or the full length light chain sequence, with (a) and(b) on separate nucleic acid molecules.

The present invention also provides an expression vector comprising anucleic acid molecule as described herein. Also provided is arecombinant host cell comprising an expression vector as describedherein.

The antigen binding protein described herein may be produced in asuitable host cell. A method for the production of the antigen bindingprotein as described herein may comprise the step of culturing a hostcell as described herein and recovering the antigen binding protein. Arecombinant transformed, transfected, or transduced host cell maycomprise at least one expression cassette, whereby said expressioncassette comprises a polynucleotide encoding a heavy chain of theantigen binding protein described herein and further comprises apolynucleotide encoding a light chain of the antigen binding proteindescribed herein. Alternatively, a recombinant transformed, transfectedor transduced hot cell may comprise at least one expression cassette,whereby a first expression cassette comprises a polynucleotide encodinga heavy chain of the antigen binding protein described herein andfurther comprise a second cassette comprising a polynucleotide encodinga light chain of the antigen binding protein described herein. A stablytransformed host cell may comprise a vector comprising one or moreexpression cassettes encoding a heavy chain and/or a light chain of theantigen binding protein described herein. For example such host cellsmay comprise a first vector encoding the light chain and a second vectorencoding the heavy chain.

The host cell may be eukaryotic, for example mammalian. Examples of suchcell lines include CHO or NS0. The host cell may be a non-human hostcell. The host cell may be a non-embryonic host cell. The host cell maybe cultured in a culture media, for example serum-free culture media.The antigen binding protein may be secreted by the host cell into theculture media. The antigen binding protein can be purified to at least95% or greater (e.g. 98% or greater) with respect to said culture mediacontaining the antigen binding protein.

A pharmaceutical composition comprising the antigen binding protein anda pharmaceutically acceptable carrier is also provided by the presentinvention. A kit-of-parts comprising the pharmaceutical compositiontogether with instructions for use is further provided. For convenience,the kit-of-parts may comprise the reagents in predetermined amounts withinstructions for use.

Antibody Structures

Intact Antibodies

The light chains of antibodies from most vertebrate species can beassigned to one of two types called Kappa and Lambda based on the aminoacid sequence of the constant region. Depending on the amino acidsequence of the constant region of their heavy chains, human antibodiescan be assigned to five different classes, IgA, IgD, IgE, IgG and IgM.IgG and IgA can be further subdivided into subclasses, IgG1, IgG2, IgG3and IgG4; and IgA1 and IgA2. Species variants exist with mouse and rathaving at least IgG2a, IgG2b.

The more conserved portions of the variable region are called Frameworkregions (FR). The variable domains of intact heavy and light chains eachcomprise four FR connected by three CDRs. The CDRs in each chain areheld together in close proximity by the FR regions and with the CDRsfrom the other chain contribute to the formation of the antigen bindingsite of antibodies.

The constant regions are not directly involved in the binding of theantibody to the antigen but exhibit various effector functions such asparticipation in antibody dependent cell-mediated cytotoxicity (ADCC),phagocytosis via binding to Fcγ receptor, half-life/clearance rate vianeonatal Fc receptor (FcRn) and complement dependent cytotoxicity viathe C1q component of the complement cascade.

The human IgG2 constant region has been reported to essentially lack theability to activate complement by the classical pathway or to mediateantibody-dependent cellular cytotoxicity. The IgG4 constant region hasbeen reported to lack the ability to activate complement by theclassical pathway and mediates antibody-dependent cellular cytotoxicityonly weakly. Antibodies essentially lacking these effector functions maybe termed ‘non-lytic’ antibodies. It may be desirable to reduce theeffector function of the antibody according to the invention, optionallyto the extent that the antibody has essentially no effector function. Inan embodiment, the antibody according to the invention is non-lytic. Inan embodiment, the antibody according to the invention has essentiallyno effector function. The antibody may, or may not, be conjugated toanother molecule, for instance a molecule intended to modify theeffector function such as a cytotoxic moiety or a radioactive moiety. Inan embodiment, the antibody is not conjugated to another molecule suchas a radiolabel or cytotoxic molecule. In this embodiment, the antibodyachieves its functional effect by blocking a natural biologicalinteraction, rather than by a direct cell-killing effect.

Human Antibodies

Human antibodies may be produced by a number of methods known to thoseof skill in the art. Human antibodies can be made by the hybridomamethod using human myeloma or mouse-human heteromyeloma cells lines seeKozbor (1984) J. Immunol. 133, 3001, and Brodeur, Monoclonal AntibodyProduction Techniques and Applications, 51-63 (Marcel Dekker Inc, 1987).Alternative methods include the use of phage libraries or transgenicmice both of which utilize human variable region repertories (see Winter(1994) Annu. Rev. Immunol 12: 433-455; Green (1999) J. Immunol. Methods231: 11-23).

Several strains of transgenic mice are now available wherein their mouseimmunoglobulin loci has been replaced with human immunoglobulin genesegments (see Tomizuka (2000) PNAS 97: 722-727; Fishwild (1996) NatureBiotechnol. 14: 845-851; Mendez (1997) Nature Genetics, 15: 146-156).Upon antigen challenge such mice are capable of producing a repertoireof human antibodies from which antibodies of interest can be selected.

Phage display technology can be used to produce human antigen bindingproteins (and fragments thereof), see McCafferty (1990) Nature 348:552-553 and Griffiths et al. (1994) EMBO 13: 3245-3260.

The technique of affinity maturation (Marks Bio/technol (1992) 10:779-783) may be used to improve binding affinity wherein the affinity ofthe primary human antibody is improved by sequentially replacing the Hand L chain variable regions with naturally occurring variants andselecting on the basis of improved binding affinities. Variants of thistechnique such as “epitope imprinting” are now also available, see forexample WO 93/06213; Waterhouse (1993) Nucl. Acids Res. 21: 2265-2266.

Chimeric and Humanised Antibodies

Chimeric antibodies are typically produced using recombinant DNAmethods. DNA encoding the antibodies (e.g. cDNA) is isolated andsequenced using conventional procedures (e.g. by using oligonucleotideprobes that are capable of binding specifically to genes encoding the Hand L chains of the antibody. Hybridoma cells serve as a typical sourceof such DNA. Once isolated, the DNA is placed into expression vectorswhich are then transfected into host cells such as E. coli, COS cells,CHO cells or myeloma cells that do not otherwise produce immunoglobulinprotein to obtain synthesis of the antibody. The DNA may be modified bysubstituting the coding sequence for human L and H chains for thecorresponding non-human (e.g. murine) H and L constant regions, see forexample Morrison (1984) PNAS 81: 6851.

A large decrease in immunogenicity can be achieved by grafting only theCDRs of a non-human (e.g. murine) antibodies (“donor” antibodies) ontohuman framework (“acceptor framework”) and constant regions to generatehumanised antibodies (see Jones et al. (1986) Nature 321: 522-525; andVerhoeyen et al. (1988) Science 239: 1534-1536). However, CDR graftingper se may not result in the complete retention of antigen-bindingproperties and it is frequently found that some framework residues(sometimes referred to as “back mutations”) of the donor antibody needto be preserved in the humanised molecule if significant antigen-bindingaffinity is to be recovered (see Queen et al. (1989) PNAS 86:10,029-10,033: Co et al. (1991) Nature 351: 501-502). In this case,human variable regions showing the greatest sequence homology to thenon-human donor antibody are chosen from a database in order to providethe human framework (FR). The selection of human FRs can be made eitherfrom human consensus or individual human antibodies. Where necessary,key residues from the donor antibody can be substituted into the humanacceptor framework to preserve CDR conformations. Computer modelling ofthe antibody maybe used to help identify such structurally importantresidues, see WO 99/48523.

Alternatively, humanisation maybe achieved by a process of “veneering”.A statistical analysis of unique human and murine immunoglobulin heavyand light chain variable regions revealed that the precise patterns ofexposed residues are different in human and murine antibodies, and mostindividual surface positions have a strong preference for a small numberof different residues (see Padlan et al. (1991) Mol. Immunol. 28:489-498; and Pedersen et al. (1994) J. Mol. Biol. 235: 959-973).Therefore it is possible to reduce the immunogenicity of a non-human Fvby replacing exposed residues in its, framework regions that differ fromthose usually found in human antibodies. Because protein antigenicitymay be correlated with surface accessibility, replacement of the surfaceresidues may be sufficient to render the mouse variable region“invisible” to the human immune system (see also Mark et al. (1994) inHandbook of Experimental Pharmacology Vol. 113: The pharmacology ofMonoclonal Antibodies, Springer-Verlag, 105-134). This procedure ofhumanisation is referred to as “veneering” because only the surface ofthe antibody is altered, the supporting residues remain undisturbed.Further alternative approaches include that set out in WO04/006955 andthe procedure of Humaneering™ (Kalobios) which makes use of bacterialexpression systems and produces antibodies that are close to humangermline in sequence (Alfenito-M Advancing Protein Therapeutics January2007, San Diego, Calif.).

Bispecific Antigen Binding Proteins

A bispecific antigen binding protein is an antigen binding proteinhaving binding specificities for at least two different epitopes.Methods of making such antigen binding proteins are known in the art.Traditionally, the recombinant production of bispecific antigen bindingproteins is based on the co-expression of two immunoglobulin H chain-Lchain pairs, where the two H chains have different bindingspecificities, see Millstein et al. (1983) Nature 305: 537-539; WO93/08829; and Traunecker et al. (1991) EMBO 10: 3655-3659. Because ofthe random assortment of H and L chains, a potential mixture of tendifferent antibody structures are produced of which only one has thedesired binding specificity. An alternative approach involves fusing thevariable domains with the desired binding specificities to heavy chainconstant region comprising at least part of the hinge region, CH2 andCH3 regions. The CH1 region containing the site necessary for lightchain binding may be present in at least one of the fusions. DNAencoding these fusions, and if desired the L chain are inserted intoseparate expression vectors and are then co-transfected into a suitablehost organism. It is possible though to insert the coding sequences fortwo or all three chains into one expression vector. In one approach, thebispecific antibody is composed of a H chain with a first bindingspecificity in one arm and a H-L chain pair, providing a second bindingspecificity in the other arm, see WO 94/04690. Also see Suresh et al.(1986) Methods in Enzymology 121: 210.

Antigen Binding Fragments

Fragments lacking the constant region lack the ability to activatecomplement by the classical pathway or to mediate antibody-dependentcellular cytotoxicity. Traditionally such fragments are produced by theproteolytic digestion of intact antibodies by e.g. papain digestion (seefor example, WO 94/29348) but may be produced directly fromrecombinantly transformed host cells. For the production of ScFv, seeBird et al. (1988) Science 242: 423-426. In addition, antigen bindingfragments may be produced using a variety of engineering techniques asdescribed below.

Fv fragments appear to have lower interaction energy of their two chainsthan Fab fragments. To stabilise the association of the V_(H) and V_(L)domains, they have been linked with peptides (Bird et al. (1988) Science242: 423-426; Huston et al. (1988) PNAS 85(16): 5879-5883), disulphidebridges (Glockshuber et al. (1990) Biochemistry 29: 1362-1367) and “knobin hole” mutations (Zhu et al. (1997) Protein Sci., 6: 781-788). ScFvfragments can be produced by methods well known to those skilled in theart, see Whitlow et al. (1991) Methods Companion Methods Enzymol, 2:97-105 and Huston et al. (1993) Int. Rev. Immunol 10: 195-217. ScFv maybe produced in bacterial cells such as E. coli or in eukaryotic cells.One disadvantage of ScFv is the monovalency of the product, whichprecludes an increased avidity due to polyvalent binding, and theirshort half-life. Attempts to overcome these problems include bivalent(ScFv′)₂ produced from ScFv containing an additional C-terminal cysteineby chemical coupling (Adams et al. (1993) Can. Res 53: 4026-4034; andMcCartney et al. (1995) Protein Eng. 8: 301-314) or by spontaneoussite-specific dimerisation of ScFv containing an unpaired C-terminalcysteine residue (see Kipriyanov et al. (1995) Cell. Biophys 26:187-204). Alternatively, ScFv can be forced to form multimers byshortening the peptide linker to 3 to 12 residues to form “diabodies”,see Holliger et al. (1993) PNAS 90: 6444-6448. Reducing the linker stillfurther can result in ScFv trimers (“triabodies”, see Kortt et al.(1997) Protein Eng 10: 423-433) and tetramers (“tetrabodies”, see LeGall et al. (1999) FEBS Lett, 453: 164-168). Construction of bivalentScFv molecules can also be achieved by genetic fusion with proteindimerising motifs to form “miniantibodies” (see Pack et al. (1992)Biochemistry 31: 1579-1584) and “minibodies” (see Hu et al. (1996)Cancer Res. 56: 3055-3061). ScFv-Sc-Fv tandems ((ScFv)₂) may also beproduced by linking two ScFv units by a third peptide linker, see Kuruczet al. (1995) J. Immol. 154: 4576-4582. Bispecific diabodies can beproduced through the noncovalent association of two single chain fusionproducts consisting of V_(H) domain from one antibody connected by ashort linker to the V_(L) domain of another antibody, see Kipriyanov etal. (1998) Int. J. Can 77: 763-772. The stability of such bispecificdiabodies can be enhanced by the introduction of disulphide bridges or“knob in hole” mutations as described supra or by the formation ofsingle chain diabodies (ScDb) wherein two hybrid ScFv fragments areconnected through a peptide linker see Kontermann et al. (1999) J.Immunol. Methods 226:179-188. Tetravalent bispecific molecules areavailable by e.g. fusing a ScFv fragment to the CH3 domain of an IgGmolecule or to a Fab fragment through the hinge region, see Coloma etal. (1997) Nature Biotechnol. 15: 159-163. Alternatively, tetravalentbispecific molecules have been created by the fusion of bispecificsingle chain diabodies (see Alt et al. (1999) FEBS Lett 454: 90-94.Smaller tetravalent bispecific molecules can also be formed by thedimerization of either ScFv-ScFv tandems with a linker containing ahelix-loop-helix motif (DiBi miniantibodies, see Muller et al. (1998)FEBS Lett 432: 45-49) or a single chain molecule comprising fourantibody variable domains (V_(H) and V_(L)) in an orientation preventingintramolecular pairing (tandem diabody, see Kipriyanov et al. (1999) J.Mol. Biol. 293: 41-56). Bispecific F(ab′)₂ fragments can be created bychemical coupling of Fab′ fragments or by heterodimerization throughleucine zippers (see Shalaby et al. (1992) J. Exp. Med. 175: 217-225;and Kostelny et al. (1992), J. Immunol. 148: 1547-1553). Also availableare isolated V_(H) and V_(L) domains (Domantis plc), see U.S. Pat. No.6,248,516; U.S. Pat. No. 6,291,158; and U.S. Pat. No. 6,172,197.

Heteroconjugate Antibodies

Heteroconjugate antibodies are composed of two covalently joinedantibodies formed using any convenient cross-linking methods. See, forexample, U.S. Pat. No. 4,676,980.

Other Modifications

The antigen binding proteins of the present invention may comprise othermodifications to enhance or change their effector functions. The term“Effector Function” as used herein is meant to refer to one or more ofAntibody dependant cell mediated cytotoxic activity (ADCC),Complement-dependant cytotoxic activity (CDC) mediated responses,Fc-mediated phagocytosis and antibody recycling via the FcRn receptor.For IgG antibodies, effector functionalities including ADCC and ADCP aremediated by the interaction of the heavy chain constant region with afamily of Fcγ receptors present on the surface of immune cells. Inhumans these include FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16).Interaction between the antigen binding protein bound to antigen and theformation of the Fc/Fcγ complex induces a range of effects includingcytotoxicity, immune cell activation, phagocytosis and release ofinflammatory cytokines.

The interaction between the constant region of an antigen bindingprotein and various Fc receptors (FcR) is believed to mediate theeffector functions of the antigen binding protein. Significantbiological effects can be a consequence of effector functionality, inparticular, antibody-dependent cellular cytotoxicity (ADCC), fixation ofcomplement (complement dependent cytotoxicity or CDC), andhalf-life/clearance of the antigen binding protein. Usually, the abilityto mediate effector function requires binding of the antigen bindingprotein to an antigen and not all antigen binding proteins will mediateevery effector function.

Effector function can be measured in a number of ways including forexample via binding of the FcγRIII to Natural Killer cells or via FcγRIto monocytes/macrophages to measure for ADCC effector function. Forexample an antigen binding protein of the present invention can beassessed for ADCC effector function in a Natural Killer cell assay.Examples of such assays can be found in Shields et al, 2001 The Journalof Biological Chemistry, Vol. 276, p6591-6604; Chappel et al, 1993 TheJournal of Biological Chemistry, Vol 268, p25124-25131; Lazar et al,2006 PNAS, 103; 4005-4010.

Examples of assays to determine CDC function include that described in1995 J Imm Meth 184:29-38.

Some isotypes of human constant regions, in particular IgG4 and IgG2isotypes, essentially lack the functions of a) activation of complementby the classical pathway; and b) antibody-dependent cellularcytotoxicity. Various modifications to the heavy chain constant regionof antigen binding proteins may be carried out depending on the desiredeffector property. IgG1 constant regions containing specific mutationshave separately been described to reduce binding to Fc receptors andtherefore reduce ADCC and CDC (Duncan et al. Nature 1988, 332; 563-564;Lund et al. J. Immunol. 1991, 147; 2657-2662; Chappel et al. PNAS 1991,88; 9036-9040; Burton and Woof, Adv. Immunol. 1992, 51; 1-84; Morgan etal., Immunology 1995, 86; 319-324; Hezareh et al., J. Virol. 2001, 75(24); 12161-12168).

Various modifications to the Fc region of antibodies may be carried outdepending on the desired property. For example, specific mutations inthe Fc region to render an otherwise lytic antibody, non-lytic aredetailed in EP 0629 240 and EP 0307 434 or one may incorporate a salvagereceptor binding epitope into the antibody to increase serum half lifesee U.S. Pat. No. 5,739,277. Human Fcγ receptors include FcγR (I),FcγRIIa, FcγRIIb, FcγRIIIa and neonatal FcRn. Shields et al. (2001) J.Biol. Chem. 276: 6591-6604 demonstrated that a common set of IgG1residues is involved in binding all FcγRs, while FcγRII and FcγRIIIutilize distinct sites outside of this common set. One group of IgG1residues reduced binding to all FcγRs when altered to alanine: Pro-238,Asp-265, Asp-270, Asn-297 and Pro-239. All are in the IgG CH2 domain andclustered near the hinge joining CH1 and CH2. While FcγRI utilizes onlythe common set of IgG1 residues for binding, FcγRII and FcγRIII interactwith distinct residues in addition to the common set. Alteration of someresidues reduced binding only to FcγRII (e.g. Arg-292) or FcγRIII (e.g.Glu-293). Some variants showed improved binding to FcγRII or FcγRIII butdid not affect binding to the other receptor (e.g. Ser-267Ala improvedbinding to FcγRII but binding to FcγRIII was unaffected). Other variantsexhibited improved binding to FcγRII or FcγRIII with reduction inbinding to the other receptor (e.g. Ser-298Ala improved binding toFcγRIII and reduced binding to FcγRII). For FcγRIIIa, the best bindingIgG1 variants had combined alanine substitutions at Ser-298, Glu-333 andLys-334. The neonatal FcRn receptor is believed to be involved in bothantibody clearance and the transcytosis across tissues (see Junghans(1997) Immunol. Res 16: 29-57; and Ghetie et al. (2000) Annu. Rev.Immunol. 18: 739-766). Human IgG1 residues determined to interactdirectly with human FcRn includes Ile253, Ser254, Lys288, Thr307,Gln311, Asn434 and His435. Substitutions at any of the positionsdescribed in this section may enable increased serum half-life and/oraltered effector properties of the antibodies.

Other modifications include glycosylation variants of the antibodies.Glycosylation of antibodies at conserved positions in their constantregions is known to have a profound effect on antibody function,particularly effector functioning such as those described above, see forexample, Boyd et al. (1996) Mol. Immunol. 32: 1311-1318. Glycosylationvariants of the antibodies or antigen binding fragments thereof whereinone or more carbohydrate moiety is added, substituted, deleted ormodified are contemplated. Introduction of an asparagine-X-serine orasparagine-X-threonine motif creates a potential site for enzymaticattachment of carbohydrate moieties and may therefore be used tomanipulate the glycosylation of an antibody. In Raju et al. (2001)Biochemistry 40: 8868-8876 the terminal sialyation of a TNFR-IgGimmunoadhesin was increased through a process of regalactosylationand/or resialylation using beta-1, 4-galactosyltransferace and/or alpha,2,3 sialyltransferase. Increasing the terminal sialylation is believedto increase the half-life of the immunoglobulin. Antibodies, in commonwith most glycoproteins, are typically produced as a mixture ofglycoforms. This mixture is particularly apparent when antibodies areproduced in eukaryotic, particularly mammalian cells. A variety ofmethods have been developed to manufacture defined glycoforms, see Zhanget al. (2004) Science 303: 371: Sears et al. (2001) Science 291: 2344;Wacker et al. (2002) Science 298: 1790; Davis et al. (2002) Chem. Rev.102: 579; Hang et al. (2001) Acc. Chem. Res 34: 727. The antibodies (forexample of the IgG isotype, e.g. IgG1) as herein described may comprisea defined number (e.g. 7 or less, for example 5 or less, such as two ora single) of glycoform(s).

The antibodies may or may not be coupled to a non-proteinaeous polymersuch as polyethylene glycol (PEG), polypropylene glycol orpolyoxyalkylene. Conjugation of proteins to PEG is an establishedtechnique for increasing half-life of proteins, as well as reducingantigenicity and immunogenicity of proteins. The use of PEGylation withdifferent molecular weights and styles (linear or branched) has beeninvestigated with intact antibodies as well as Fab′ fragments, seeKoumenis et al. (2000) Int. J. Pharmaceut. 198: 83-95.

Production Methods

Antigen binding proteins may be produced in transgenic organisms such asgoats (see Pollock et al. (1999) J. Immunol. Methods 231: 147-157),chickens (see Morrow (2000) Genet. Eng. News 20:1-55, mice (see Pollocket al.) or plants (see Doran (2000) Curr. Opinion Biotechnol. 11:199-204; Ma (1998) Nat. Med. 4: 601-606; Baez et al. (2000) BioPharm 13:50-54; Stoger et al. (2000) Plant Mol. Biol. 42: 583-590).

Antigen binding proteins may also be produced by chemical synthesis.However, antigen binding proteins are typically produced usingrecombinant cell culturing technology well known to those skilled in theart. A polynucleotide encoding the antigen binding protein is isolatedand inserted into a replicable vector such as a plasmid for furthercloning (amplification) or expression. One expression system is aglutamate synthetase system (such as sold by Lonza Biologics),particularly where the host cell is CHO or NS0. Polynucleotide encodingthe antigen binding protein is readily isolated and sequenced usingconventional procedures (e.g. oligonucleotide probes). Vectors that maybe used include plasmid, virus, phage, transposons, minichromosomes ofwhich plasmids are typically used. Generally such vectors furtherinclude a signal sequence, origin of replication, one or more markergenes, an enhancer element, a promoter and transcription terminationsequences operably linked to the antigen binding protein polynucleotideso as to facilitate expression. Polynucleotide encoding the light andheavy chains may be inserted into separate vectors and introduced (forexample by transformation, transfection, electroporation ortransduction) into the same host cell concurrently or sequentially or,if desired both the heavy chain and light chain can be inserted into thesame vector prior to said introduction.

Codon optimisation may be used with the intent that the total level ofprotein produced by the host cell is greater when transfected with thecodon-optimised gene in comparison with the level when transfected withthe wild-type sequence. Several methods have been published (Nakamura etal. (1996) Nucleic Acids Research 24: 214-215; WO98/34640; WO97/11086).Due to the redundancy of the genetic code, alternative polynucleotidesto those disclosed herein (particularly those codon optimised forexpression in a given host cell) may also encode the antigen bindingproteins described herein. The codon usage of the antigen bindingprotein of this invention thereof can be modified to accommodate codonbias of the host cell such to augment transcript and/or product yield(eg Hoekema et al Mol Cell Biol 1987 7(8): 2914-24). The choice ofcodons may be based upon suitable compatibility with the host cell usedfor expression.

Signal Sequences

Antigen binding proteins may be produced as a fusion protein with aheterologous signal sequence having a specific cleavage site at theN-terminus of the mature protein. The signal sequence should berecognised and processed by the host cell. For prokaryotic host cells,the signal sequence may be for example an alkaline phosphatase,penicillinase, or heat stable enterotoxin II leaders. For yeastsecretion the signal sequences may be for example a yeast invertaseleader, α factor leader or acid phosphatase leaders see e.g. WO90/13646.In mammalian cell systems, viral secretory leaders such as herpessimplex gD signal and a native immunoglobulin signal sequence may besuitable. Typically the signal sequence is ligated in reading frame toDNA encoding the antigen binding protein.

Origin of Replication

Origin of replications are well known in the art with pBR322 suitablefor most gram-negative bacteria, 2μ plasmid for most yeast and variousviral origins such as SV40, polyoma, adenovirus, VSV or BPV for mostmammalian cells. Generally the origin of replication component is notneeded for mammalian expression vectors but the SV40 may be used sinceit contains the early promoter.

Selection Marker

Typical selection genes encode proteins that (a) confer resistance toantibiotics or other toxins e.g. ampicillin, neomycin, methotrexate ortetracycline or (b) complement auxiotrophic deficiencies or supplynutrients not available in the complex media or (c) combinations ofboth. The selection scheme may involve arresting growth of the hostcell. Cells, which have been successfully transformed with the genesencoding the antigen binding protein, survive due to e.g. drugresistance conferred by the co-delivered selection marker. One exampleis the DHFR selection marker wherein transformants are cultured in thepresence of methotrexate. Cells can be cultured in the presence ofincreasing amounts of methotrexate to amplify the copy number of theexogenous gene of interest. CHO cells are a particularly useful cellline for the DHFR selection. A further example is the glutamatesynthetase expression system (Lonza Biologics). An example of aselection gene for use in yeast is the trp1 gene, see Stinchcomb et al.(1979) Nature 282: 38.

Promoters

Suitable promoters for expressing antigen binding proteins are operablylinked to DNA/polynucleotide encoding the antigen binding protein.Promoters for prokaryotic hosts include phoA promoter, beta-lactamaseand lactose promoter systems, alkaline phosphatase, tryptophan andhybrid promoters such as Tac. Promoters suitable for expression in yeastcells include 3-phosphoglycerate kinase or other glycolytic enzymes e.g.enolase, glyceralderhyde 3 phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose 6 phosphate isomerase,3-phosphoglycerate mutase and glucokinase. Inducible yeast promotersinclude alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,metallothionein and enzymes responsible for nitrogen metabolism ormaltose/galactose utilization.

Promoters for expression in mammalian cell systems include viralpromoters such as polyoma, fowlpox and adenoviruses (e.g. adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus (inparticular the immediate early gene promoter), retrovirus, hepatitis Bvirus, actin, rous sarcoma virus (RSV) promoter and the early or lateSimian virus 40. Of course the choice of promoter is based upon suitablecompatibility with the host cell used for expression. A first plasmidmay comprise a RSV and/or SV40 and/or CMV promoter, DNA encoding lightchain variable region (V_(L)), κC region together with neomycin andampicillin resistance selection markers and a second plasmid comprisinga RSV or SV40 promoter, DNA encoding the heavy chain variable region(V_(H)), DNA encoding the γ1 constant region, DHFR and ampicillinresistance markers.

Enhancer Element

Where appropriate, e.g. for expression in higher eukaryotes, an enhancerelement operably linked to the promoter element in a vector may be used.Mammalian enhancer sequences include enhancer elements from globin,elastase, albumin, fetoprotein and insulin. Alternatively, one may usean enhancer element from a eukaroytic cell virus such as SV40 enhancer(at bp 100-270), cytomegalovirus early promoter enhancer, polymaenhancer, baculoviral enhancer or murine IgG2a locus (see WO04/009823).The enhancer may be located on the vector at a site upstream to thepromoter. Alternatively, the enhancer may be located elsewhere, forexample within the untranslated region or downstream of thepolyadenylation signal. The choice and positioning of enhancer may bebased upon suitable compatibility with the host cell used forexpression.

Polyadenylation/Termination

In eukaryotic systems, polyadenylation signals are operably linked toDNA/polynucleotide encoding the antigen binding protein. Such signalsare typically placed 3′ of the open reading frame. In mammalian systems,non-limiting examples include signals derived from growth hormones,elongation factor-1 alpha and viral (eg SV40) genes or retroviral longterminal repeats. In yeast systems non-limiting examples ofpolydenylation/termination signals include those derived from thephosphoglycerate kinase (PGK) and the alcohol dehydrogenase 1 (ADH)genes. In prokaryotic system polyadenylation signals are typically notrequired and it is instead usual to employ shorter and more definedterminator sequences. The choice of polyadenylation/terminationsequences may be based upon suitable compatibility with the host cellused for expression.

Other Methods/Elements for Enhanced Yields

In addition to the above, other features that can be employed to enhanceyields include chromatin remodelling elements, introns and host-cellspecific codon modification.

Host Cells

Suitable host cells for cloning or expressing vectors encoding antigenbinding proteins are prokaroytic, yeast or higher eukaryotic cells.Suitable prokaryotic cells include eubacteria e.g. enterobacteriaceaesuch as Escherichia e.g. E. coli (for example ATCC 31,446; 31,537;27,325), Enterobacter, Erwinia, Klebsiella Proteus, Salmonella e.g.Salmonella typhimurium, Serratia e.g. Serratia marcescans and Shigellaas well as Bacilli such as B. subtilis and B. licheniformis (see DD 266710), Pseudomonas such as P. aeruginosa and Streptomyces. Of the yeasthost cells, Saccharomyces cerevisiae, Schizosaccharomyces pombe,Kluyveromyces (e.g. ATCC 16,045; 12,424; 24178; 56,500), yarrowia(EP402, 226), Pichia pastoris (EP 183 070, see also Peng et al. (2004)J. Biotechnol. 108: 185-192), Candida, Trichoderma reesia (EP 244 234),Penicillin, Tolypocladium and Aspergillus hosts such as A. nidulans andA. niger are also contemplated.

Higher eukaryotic host cells include mammalian cells such as COS-1 (ATCCNo. CRL 1650) COS-7 (ATCC CRL 1651), human embryonic kidney line 293,baby hamster kidney cells (BHK) (ATCC CRL.1632), BHK570 (ATCC NO: CRL10314), 293 (ATCC NO. CRL 1573), Chinese hamster ovary cells CHO (e.g.CHO-K1, ATCC NO: CCL 61, DHFR-CHO cell line such as DG44 (see Urlaub etal. (1986) Somatic Cell Mol. Genet. 12: 555-556), particularly those CHOcell lines adapted for suspension culture, mouse sertoli cells, monkeykidney cells, African green monkey kidney cells (ATCC CRL-1587), HELAcells, canine kidney cells (ATCC CCL 34), human lung cells (ATCC CCL75), Hep G2 and myeloma or lymphoma cells e.g. NS0 (see U.S. Pat. No.5,807,715), Sp2/0, Y0.

Such host cells may also be further engineered or adapted to modifyquality, function and/or yield of the antigen binding protein.Non-limiting examples include expression of specific modifying (e.g.glycosylation) enzymes and protein folding chaperones.

Cell Culturing Methods

Host cells transformed with vectors encoding antigen binding proteinsmay be cultured by any method known to those skilled in the art. Hostcells may be cultured in spinner flasks, roller bottles or hollow fibresystems but for large scale production that stirred tank reactors areused particularly for suspension cultures. The stirred tankers may beadapted for aeration using e.g. spargers, baffles or low shearimpellers. For bubble columns and airlift reactors direct aeration withair or oxygen bubbles maybe used. Where the host cells are cultured in aserum free culture media, the media is supplemented with a cellprotective agent such as pluronic F-68 to help prevent cell damage as aresult of the aeration process. Depending on the host cellcharacteristics, either microcarriers maybe used as growth substratesfor anchorage dependent cell lines or the cells maybe adapted tosuspension culture (which is typical). The culturing of host cells,particularly invertebrate host cells may utilise a variety ofoperational modes such as fed-batch, repeated batch processing (seeDrapeau et al. (1994) Cytotechnology 15: 103-109), extended batchprocess or perfusion culture. Although recombinantly transformedmammalian host cells may be cultured in serum-containing media such asfetal calf serum (FCS), for example such host cells are cultured insynthetic serum—free media such as disclosed in Keen et al. (1995)Cytotechnology 17: 153-163, or commercially available media such asProCHO-CDM or UltraCHO™ (Cambrex N.J., USA), supplemented wherenecessary with an energy source such as glucose and synthetic growthfactors such as recombinant insulin. The serum-free culturing of hostcells may require that those cells are adapted to grow in serum freeconditions. One adaptation approach is to culture such host cells inserum containing media and repeatedly exchange 80% of the culture mediumfor the serum-free media so that the host cells learn to adapt in serumfree conditions (see e.g. Scharfenberg et al. (1995) in Animal CellTechnology: Developments towards the 21st century (Beuvery et al. eds,619-623, Kluwer Academic publishers).

Antigen binding proteins secreted into the media may be recovered andpurified using a variety of techniques to provide a degree ofpurification suitable for the intended use. For example the use ofantigen binding proteins for the treatment of human patients typicallymandates at least 95% purity, more typically 98% or 99% or greaterpurity (compared to the crude culture medium). Cell debris from theculture media is typically removed using centrifugation followed by aclarification step of the supernatant using e.g. microfiltration,ultrafiltration and/or depth filtration. A variety of other techniquessuch as dialysis and gel electrophoresis and chromatographic techniquessuch as hydroxyapatite (HA), affinity chromatography (optionallyinvolving an affinity tagging system such as polyhistidine) and/orhydrophobic interaction chromatography (HIC, see U.S. Pat. No.5,429,746) are available. The antibodies, following variousclarification steps, can be captured using Protein A or G affinitychromatography. Further chromatography steps can follow such as ionexchange and/or HA chromatography, anion or cation exchange, sizeexclusion chromatography and ammonium sulphate precipitation. Variousvirus removal steps may also be employed (e.g. nanofiltration using e.g.a DV-20 filter). Following these various steps, a purified (for examplea monoclonal) preparation comprising at least 75 mg/ml or greater, or100 mg/ml or greater, of the antigen binding protein is provided. Suchpreparations are substantially free of aggregated forms of antigenbinding proteins.

Bacterial systems may be used for the expression of antigen bindingfragments. Such fragments can be localised intracellularly, within theperiplasm or secreted extracellularly. Insoluble proteins can beextracted and refolded to form active proteins according to methodsknown to those skilled in the art, see Sanchez et al. (1999) J.Biotechnol. 72: 13-20; and Cupit et al. (1999) Lett Appl Microbiol 29:273-277.

Pharmaceutical Compositions

The terms diseases, disorders and conditions are used interchangeably.Purified preparations of an antigen binding protein as described hereinmay be incorporated into pharmaceutical compositions for use in thetreatment of the human diseases described herein. The pharmaceuticalcomposition can be used in the treatment of diseases where IL-7contributes to the disease or where inhibition/neutralisation ofIL-7R-mediated signalling will be beneficial. The pharmaceuticalcomposition comprises a therapeutically effective amount of the antigenbinding protein described herein.

The pharmaceutical preparation may comprise an antigen binding proteinin combination with a pharmaceutically acceptable carrier. The antigenbinding protein may be administered alone, or as part of apharmaceutical composition.

Typically such compositions comprise a pharmaceutically acceptablecarrier as known and called for by acceptable pharmaceutical practice,see e.g. Remingtons Pharmaceutical Sciences, 16th edition (1980) MackPublishing Co. Examples of such carriers include sterilised carrierssuch as saline, Ringers solution or dextrose solution, optionallybuffered with suitable buffers to a pH within a range of 5 to 8.

Pharmaceutical compositions may be administered by injection orcontinuous infusion (e.g. intravenous, intraperitoneal, intradermal,subcutaneous, intramuscular or intraportal). Such compositions aresuitably free of visible particulate matter. Pharmaceutical compositionsmay comprise between 1 mg to 10 g of antigen binding protein, forexample between 5 mg and 1 g of antigen binding protein. Alternatively,the composition may comprise between 5 mg and 500 mg, for examplebetween 5 mg and 50 mg.

Methods for the preparation of such pharmaceutical compositions are wellknown to those skilled in the art. Pharmaceutical compositions maycomprise between 1 mg to 10 g of antigen binding protein in unit dosageform, optionally together with instructions for use. Pharmaceuticalcompositions may be lyophilised (freeze dried) for reconstitution priorto administration according to methods well known or apparent to thoseskilled in the art. Where antibodies have an IgG1 isotype, a chelator ofcopper, such as citrate (e.g. sodium citrate) or EDTA or histidine, maybe added to the pharmaceutical composition to reduce the degree ofcopper-mediated degradation of antibodies of this isotype, seeEP0612251. Pharmaceutical compositions may also comprise a solubilisersuch as arginine base, a detergent/anti-aggregation agent such aspolysorbate 80, and an inert gas such as nitrogen to replace vialheadspace oxygen.

Effective doses and treatment regimes for administering the antigenbinding protein are generally determined empirically and may bedependent on factors such as the age, weight and health status of thepatient and disease or disorder to be treated. Such factors are withinthe purview of the attending physician. Guidance in selectingappropriate doses may be found in e.g. Smith et al (1977) Antibodies inhuman diagnosis and therapy, Raven Press, New York. Thus the antigenbinding protein of the invention may be administered at atherapeutically effective amount.

The dosage of antigen binding protein administered to a subject isgenerally between 1 μg/kg to 150 mg/kg, between 0.1 mg/kg and 100 mg/kg,between 0.5 mg/kg and 50 mg/kg, between 1 and 25 mg/kg or between 1 and10 mg/kg of the subject's body weight. For example, the dose may be 10mg/kg, 30 mg/kg, or 60 mg/kg. The antigen binding protein may beadministered parenterally, for example subcutaneously, intravenously orintramuscularly.

If desired, the effective daily dose of a therapeutic composition may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals, optionally, in unitdosage forms. For example, the dose may be administered subcutaneously,once every 14 or 28 days in the form of multiple sub-doses on each dayof administration.

The administration of a dose may be by intravenous infusion, typicallyover a period of from 15 minutes to 24 hours, such as from 2 to 12hours, or from 2 to 6 hours. This may result in reduced toxic sideeffects.

The administration of a dose may be repeated one or more times asnecessary, for example, three times daily, once every day, once every 2days, once a week, once a fortnight, once a month, once every 3 months,once every 6 months, or once every 12 months. The antigen bindingproteins may be administered by maintenance therapy, for example once aweek for a period of 6 months or more. The antigen binding proteins maybe administered by intermittent therapy, for example for a period of 3to 6 months and then no dose for 3 to 6 months, followed byadministration of antigen binding proteins again for 3 to 6 months, andso on in a cycle.

The dosage may be determined or adjusted by measuring the amount ofIL-17 in a biological sample. Other means of determining or adjustingdosage may be utilized, including but not limited to biologic markers(‘biomarkers’) of pharmacology, measures of muscle mass and/or function,safety, tolerability, and therapeutic response. The antigen bindingprotein can be administered in an amount and for a duration effective todown-regulate IL-7 mediated signalling activity in the subject.

The antigen binding protein may be administered to the subject in such away as to target therapy to a particular site. For example, the antigenbinding protein may be injected locally into muscle, for exampleskeletal muscle.

The antigen binding protein may be used in combination with one or moreother therapeutically active agents, for example: immunomodulators suchas interferon beta (IFNβ-1a or IFNβ-1b) and glatiramer acetate,immunosuppresants such as cyclophosphamide, methotrexate, azathioprine,cladribine, cyclosporine and mitoxantrone, other immune therapies suchas intravenous immune globulin (IVIg), plasma replacement andsulphasalazine. The additional therapeutic may be administered as in amanner (dosage, timing, mechanism) as prescribed by a physician. In anembodiment, the additional therapeutic agent may be administeredsimultaneously or sequentially or separately from the antigen bindingprotein of the present invention. In an embodiment, the additionaltherapeutic agent and the antigen binding protein are administered suchthat their pharmacological effects on the patient overlap; in otherwords, they exert their biological effects on the patient at the sametime.

When the antigen binding protein is used in combination with othertherapeutically active agents, the individual components may beadministered either together or separately, sequentially orsimultaneously, in separate or combined pharmaceutical formulations, byany appropriate route. If administered separately or sequentially, theantigen binding protein and the therapeutically active agent(s) may beadministered in any order.

The combinations referred to above may be presented for use in the formof a single pharmaceutical formulation comprising a combination asdefined above optionally together with a pharmaceutically acceptablecarrier or excipient.

When combined in the same formulation it will be appreciated that thecomponents must be stable and compatible with each other and the othercomponents of the formulation and may be formulated for administration.When formulated separately they may be provided in any convenientformulation, for example in such a manner as known for antigen bindingproteins in the art.

When in combination with a second therapeutic agent active against thesame disease, the dose of each component may differ from that when theantigen binding protein is used alone. Appropriate doses will be readilyappreciated by those skilled in the art.

The antigen binding protein and the therapeutically active agent(s) mayact synergistically. In other words, administering the antigen bindingprotein and the therapeutically active agent(s) in combination may havea greater effect on the disease, disorder, or condition described hereinthan the sum of the effect of each alone.

The pharmaceutical composition may comprise a kit of parts of theantigen binding protein together with other medicaments, optionally withinstructions for use. For convenience, the kit may comprise the reagentsin predetermined amounts with instructions for use.

The terms “individual”, “subject” and “patient” are used hereininterchangeably. The subject is typically a human. The subject may alsobe a mammal, such as a mouse, rat or primate (e.g. a marmoset ormonkey). The subject can be a non-human animal. The antigen bindingproteins may also have veterinary use. The subject to be treated may bea farm animal for example, a cow or bull, sheep, pig, ox, goat or horseor may be a domestic animal such as a dog or cat. The animal may be anyage, or a mature adult animal.

Treatment may be therapeutic, prophylactic or preventative. The subjectmay be one who is in need thereof. Those in need of treatment mayinclude individuals already suffering from a particular medical diseasein addition to those who may develop the disease in the future.

Thus, the antigen binding protein described herein can be used forprophylactic or preventative treatment. In this case, the antigenbinding protein described herein is administered to an individual inorder to prevent or delay the onset of one or more aspects or symptomsof the disease. The subject can be asymptomatic. The subject may have agenetic predisposition to the disease. A prophylactically effectiveamount of the antigen binding protein is administered to such anindividual. A prophylactically effective amount is an amount whichprevents or delays the onset of one or more aspects or symptoms of adisease described herein.

The antigen binding protein described herein may also be used in methodsof therapy. The term “therapy” encompasses alleviation, reduction, orprevention of at least one aspect or symptom of a disease. For example,the antigen binding protein described herein may be used to ameliorateor reduce one or more aspects or symptoms of a disease described herein.

The antigen binding protein described herein is used in an effectiveamount for therapeutic, prophylactic or preventative treatment. Atherapeutically effective amount of the antigen binding proteindescribed herein is an amount effective to ameliorate or reduce one ormore aspects or symptoms of the disease. The antigen binding proteindescribed herein may also be used to treat, prevent, or cure the diseasedescribed herein.

The antigen binding protein described herein may have a generallybeneficial effect on the subject's health, for example it can increasethe subject's expected longevity.

The antigen binding protein described herein need not affect a completecure, or eradicate every symptom or manifestation of the disease toconstitute a viable therapeutic treatment. As is recognised in thepertinent field, drugs employed as therapeutic agents may reduce theseverity of a given disease state, but need not abolish everymanifestation of the disease to be regarded as useful therapeuticagents. Similarly, a prophylactically administered treatment need not becompletely effective in preventing the onset of a disease in order toconstitute a viable prophylactic agent. Simply reducing the impact of adisease (for example, by reducing the number or severity of itssymptoms, or by increasing the effectiveness of another treatment, or byproducing another beneficial effect), or reducing the likelihood thatthe disease will occur (for example by delaying the onset of thedisease) or worsen in a subject, is sufficient.

The antigen binding proteins of the present invention may be used in thetherapy of multiple sclerosis and in other autoimmune or inflammatorydiseases, particularly those in which pathogenic T_(H)17 cells areimplicated. Such diseases are associated with high levels of IL-17expression. Elevated levels of IL-17 have been reported in serum and CSFof MS patients (Matusevicius, D. et al.; Mult. Scler. 5, 101-104; 1999)and in the synovial fluid obtained from rheumatoid arthritis patients.IL-17 has also been implicated in psoriasis (Homey et al.; J. Immunol.164(12):6621-32; 2000), while Hamzaoui et al reported high levels ofIL-17 in Behcet's disease (Scand. J. Rhuematol.; 31:4, 205-210; 2002).Elevated IL-17 levels have also been observed in systemic lupuserythrematosus (SLE) (Wong et al.; Lupus 9(8):589-93; 2000).

Inhibition of IL-7 receptor mediated signalling may also be useful inthe treatment of inflammatory (non-autoimmune) diseases in whichelevated IL-17 has been implicated, such as asthma.

Accordingly, inflammatory and/or autoimmune diseases of the inventioninclude inflammatory skin diseases including psoriasis and atopicdermatitis; systemic scleroderma and sclerosis; inflammatory boweldisease (IBD); Crohn's disease; ulcerative colitis; ischemic reperfusiondisorders including surgical tissue reperfusion injury, myocardialischemic conditions such as myocardial infarction, cardiac arrest,reperfusion after cardiac surgery and constriction after percutaneoustransluminal coronary angioplasty, stroke, and abdominal aorticaneurysms; cerebral edema secondary to stroke; cranial trauma,hypovolemic shock; asphyxia; adult respiratory distress syndrome;acute-lung injury; Behcet's Disease; dermatomyositis; polymyositis;multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis;osteoarthritis; lupus nephritis; autoimmune diseases such as rheumatoidarthritis (RA), Sjorgen's syndrome, vasculitis; diseases involvingleukocyte diapedesis; central nervous system (CNS) inflammatorydisorder, multiple organ injury syndrome secondary to septicaemia ortrauma; alcoholic hepatitis; bacterial pneumonia; antigen-antibodycomplex mediated diseases including glomerulonephritis; sepsis;sarcoidosis; immunopathologic responses to tissue/organ transplantation;inflammations of the lung, including pleurisy, alveolitis, vasculitis,pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis,hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), andcystic fibrosis; psoriatic arthritis; neuromyelitis optica,Guillain-Barre syndrome (GBS), COPD, type 1 diabetes, etc.

In particular, the antagonists of the present invention may be useful inthe therapy of multiple sclerosis, in all its forms, includingneuromyelitis optica. Treatment with an antagonist of the presentinvention is predicted to be most efficacious when administered in thecontext of active inflammatory disease, i.e. when used in the treatmentof clinically isolated syndrome or relapsing forms of MS. These stagesof disease can be defined clinically and/or by imaging criteria such asgadolinium enhancement or other more sensitive techniques, and/or otheras yet undefined biomarkers of active disease. Particularly, theantagonists of the invention can be used to treat RRMS (via intravenous,subcutaneous, oral or intramuscular delivery) when the patients areentering or are in relapse. In an embodiment, the antagonist of theinvention is administered to the patient at the onset of relapse, orwithin 1 hr, 2 hrs, 3 hrs, 6 hrs, 12 hrs, 24 hrs, 2 days, 3 days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days from the onsetof relapse.

The antigen binding proteins of the invention are capable of binding toCD127. In an embodiment the antigen binding proteins of the inventionare capable of antagonising the biological effect of the IL-7 receptor.In an embodiment, the antigen binding proteins are capable ofantagonising at least one of: IL-7 receptor mediated T_(H)17 expansion,and IL-7 receptor mediated T_(H)17 survival.

The term inhibit, antagonise and neutralise are used hereinsynonymously. No term is intended to suggest the requirement of totalneutralisation; partial neutralisation—corresponding to a reduction butnot complete abolition of the biological effect—is also contemplated.

IL-7 receptor mediated T_(H)17 expansion and/or survival can be observedat a cellular level by an increase or maintenance of T_(H)17 cell count,or by an increase in the ratio of T_(H)17 cell numbers compared to thenumbers of other CD4+ T cells, or more specifically by an increase inthe ratio of T_(H)17:T_(H)1 cells, the ratio of T_(H)17:T_(reg) cells,the ratio of (T_(H)17 plus T_(H)1):T_(reg) cells, and/or the ratio ofT_(H)17:(T_(H)1 plus T_(reg)) cells.

At a molecular level, T_(H)17 expansion and/or survival can be observedby an increase in IL-17 production by a population of CD4+ T cells (orby a population of T_(H)17 cells). In an embodiment, therefore, theantigen binding proteins of the invention reduce IL-17 production by apopulation of CD4+ T cells. IL-7 receptor mediated T_(H)17 expansion andsurvival can also be observed by an increase in IFN-γ production by apopulation of CD4+ T cells (or by a population of T_(H)17 cells). Thus,in an embodiment, the antigen binding proteins of the inventionantagonise (reduce) IFN-γ production by a population of CD4+ T cells. Ata molecular level, the antigen binding proteins of the invention mayinhibit IL-7 receptor mediated STAT-5 phosphorylation.

At the molecular level, one can observe and measure the blocking effectof the antigen binding proteins of the invention by assays such asIL-7-induced P-STAT5 or Bcl-2. At the cellular level, one can observeand measure the blocking effect by assays such as Th17 secretion ofIL-17 or IFNγ. Exemplary assays are described in PCT application numberPCT/US2009/053136 (WO2010/017468).

In an exemplary pSTAT-5 assay, PBMCs are stimulated with IL-7 in thepresence and absence of a test agent. Cells are subsequently assessedquantitatively for the level of pSTAT-5, e.g. by staining for pSTAT-5(e.g. with a labelled anti-pSTAT-5 antibody, such as Alexa Fluor® 647Mouse Anti-Stat5 (pY694, BD [#612599])) followed by fluorescenceactivated cell sorting. The levels of phosphorylated STAT-5 could alsobe determined by ELISA. Those agents which reduce the level ofphosphorylated STAT-5 may be potential therapeutic candidates forautoimmune disease.

The antagonist may be capable of reducing levels of phosphorylatedSTAT-5 by at least 20%, 50%, 75%, 80%, 85%, 90%, 95% or 100% whencompared to STAT-5 levels in the absence of the antagonist, or whencompared to a negative control, or untreated cells. The antagonist mayhave an IC₅₀ of 50 μg/ml, 25 μg/ml or less, 10 μg/ml or less, 5 μg/ml orless, or 2 μg/ml or less. In an embodiment, the antagonist has an IC₅₀of less than or equal to 1 μg/ml, less than or equal to 0.75 μg/ml, lessthan or equal to 0.5 μg/ml, less than or equal to 0.25 μg/ml, or lessthan or equal to 0.1 μg/ml.

The antagonists of the invention are particularly effective ininhibiting the expansion of T_(H)17 cells. Expansion of T_(H)17 cellscan be determined in a T_(H)17 expansion assay, which comprisesstimulating a population of naïve T cells to expand in the presence andabsence of a test agent, followed by stimulating the cells to produceIL-17 and assessing the level of IL-17 produced by the cells in thepresence and absence of the test agent.

In an exemplary assay, human CD4+ T cells are differentiated intoT_(H)17 by stimulation with T cell receptor activation in the presenceof IL-1, IL-6, and IL-23. After 5 days of differentiation, CCR6+ cellsare sorted out to produce an enriched T_(H)17 population. Thispopulation is then stimulated with human IL-7 and the increase in IL-17and IFN-γ in the supernatant are determined. The ability of a testagent, such as an antigen binding fragment of the present invention toblock the interaction between the IL-7 and CD127 can be determined asthe presence of an antagonist of this interaction during the incubationperiod should prevent the expansion of the T_(H)17 cells leading to thereduction of IL-17 and IFN-γ production.

The antigen binding proteins of the invention may be capable of from 20%or more inhibition of IL-17 secretion in such an assay, versus anegative control. More typically, the antigen binding protein is capableof from 50%, from 75%, from 85% or from 90% or more inhibition of IL-17secretion versus the control. The antigen binding fragment may, in someembodiments, exhibit an IC₅₀ of less than or equal to 50 μg/ml in theassay. In other embodiments, the IC₅₀ may be less than or equal to 20μg/ml, 10 μg/ml or 5 μg/ml.

Thus, in another aspect, the invention provides a method for thetreatment of an autoimmune disease or inflammatory disorder, comprisingadministering to a patient an antigen binding protein of the inventionin an amount sufficient to reduce the T_(H)17 cell count in the patient.

In another aspect, the invention provides a method for the treatment ofan autoimmune disease in a human subject, comprising administering tothe subject an antigen binding protein in an amount sufficient to reduceIL-7 receptor mediated STAT-5 phosphorylation.

In another aspect, the present invention provides a method for treatingmultiple sclerosis in a patient comprising administering an antigenbinding protein of the invention to the patient, wherein the patient issuffering from relapsing remitting multiple sclerosis.

In another aspect, the invention provides a method of treating anautoimmune or inflammatory disease in a human subject, comprisingadministering to the subject an antigen binding protein of the inventionto the patient in an amount effective to reduce the ratio of T_(H)17cells relative to T_(H)1 cells.

In another aspect, the invention provides a method of treating anautoimmune or inflammatory disease in a human subject, comprisingadministering to the subject an antigen binding protein of the inventionto the patient in an amount effective to reduce the ratio of T_(H) cellsrelative to (Foxp3+) T_(reg) cells.

Diagnostic Methods of Use

The antigen binding proteins described herein may be used to detectCD127 in a biological sample in vitro or in vivo for diagnosticpurposes. For example, the anti-CD127 antigen binding proteins can beused to detect CD127 in cultured cells, in a tissue or in serum. Thetissue may have been first removed (for example a biopsy) from a humanor animal body. Conventional immunoassays may be employed, includingELISA, Western blot, immunohistochemistry, or immunoprecipitation.

The antigen binding proteins may be provided in a diagnostic kitcomprising one or more antigen binding proteins, a detectable label, andinstructions for use of the kit. For convenience, the kit may comprisethe reagents in predetermined amounts with instructions for use.

Gene Therapy

Nucleic acid molecules encoding the antigen binding proteins describedherein may be administered to a subject in need thereof. The nucleicacid molecule may express the CDRs in an appropriate scaffold or domain,the variable domain, or the full length antibody. The nucleic acidmolecule may be comprised in a vector which allows for expression in ahuman or animal cell. The nucleic acid molecule or vector may beformulated for administration with a pharmaceutically acceptableexcipient and/or one or more therapeutically active agents as discussedabove.

EXAMPLES 1.0 Humanization of 1A11 1.1 1A11 Cloning of the HybridomaVariable Regions

Total RNA was prepared from a cell pellets of the 1A11 hybridomas andRT-PCR performed to generate cDNA of the variable regions. The amplifiedvariable regions for heavy and light chain of each hybridoma were clonedinto a pCR2.1 cloning vector. Sequence for the heavy and light variableregions of each hybridoma was obtained. Sequence analysis predicted thepeptide sequences as follows (with the complementarity determiningregions highlighted):

A) 1A11 VH EVQLQQSGPELLKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNGVTSYNQKFKGKATLTVAKSSSTAYMELLSLTSEDSAVYYCARGDGNYWYFDVWGAGTTVTVSS (SEQ ID NO: 8) B) 1A11 VLEIVLTQSPAITAASLGQKVTITCSASSSVTYMHWYQQKSGTSPKPWIYEISKLASGVPVRFSGSGSGTSYSLTISSMEAEDAAIYYCQEWNYPYTFGGGT KLEIK (SEQ ID NO: 9)

A recombinant chimeric form of the antibody was made by fusing thevariable heavy and light regions to human IgG1Fc and kappa constantregions respectively.

1.2 1A11 Heavy Chain Humanization Strategy

Following a BLAST analysis of the human V gene germline databases, humangermline IGHV1_(—)2 which had 64% identity (including CDRs) with themouse 1A11 variable heavy chain sequence was selected as the preferredacceptor framework for humanisation. The germline V region was combinedin silico with a suitable FR4, in this case the JH6 minigene (Kabat Vol.II) based on sequence similarity. The first six residues of the JH6minigene residues preceding the WGQG motif fall within the CDR3 regionwhich is replaced by the incoming CDR from the donor antibody. Eighthumanised heavy chain variants were generated on the basis of sequencecomparison and possible impact on antibody function. Construct H0 was astraight graft of mouse CDRs from 1A11 (using the Kabat definition) intothe human acceptor framework selected above. Constructs H1 through H3are based on H0; each incorporates one additional framework mutationwhich was different in each construct; positions 71, 66 and 69respectively. H4 through H7 constructs incorporate two or three of theabove back mutations optionally with further back mutations at positions72 and 73.

1.3 1A11Heavy Chain Humanization Rationale for Framework IGHV1-2

Kabat position 66 69 71 72 73 VH1A11 K L V A K IGHV1_2 R M R D T 1A11H0R M R D T 1A11H1 R M V D T 1A11H2 K M R D T 1A11H3 R L R D T 1A11H4 K MV D T 1A11H5 K L V D T 1A11H6 K L V D K 1A11H7 K L V A K

1.4 1A11 Light Chain Humanization Strategy

Following a BLAST analysis of the human V gene germline databases, humangermline IGKV3_(—)11 which had 53% identity (including CDRs) with themouse 1A11 variable light chain sequence was selected as the preferredacceptor framework for humanisation. The germline V region was combinedin silico with a suitable FR4, in this case the J-region kappa 4minigene (Kabat Vol. II) based on sequence similarity. The first threeresidues of the JK-4 minigene residues fall within the CDR3 region whichis replaced by the incoming CDR from the donor antibody. Ten humanisedlight chain variants were generated on the basis of sequence comparisonand possible impact on antibody function. Construct L0 was a straightgraft of mouse CDRs from 1A11 (using the Kabat definition) into thehuman acceptor framework selected above. Constructs L1, L2, L4 are basedon L0, each incorporates one additional framework mutation which weredifferent in each construct; positions 47, 71 and 46 respectively.Construct L3 incorporates both of the above back mutations 47 and 71.Construct L5 incorporates three of the above back mutations 47 and 71and 46. Construct L6 through L9 are based on L5, each incorporates one,two, three and four additional framework mutations which were differentin each construct; positions 58, 45, 70 and 60 respectively.

1.5 1A11 Light Chain Rationale for Framework IGKV3-11

Kabat position 45 46 47 58 60 70 71 Vk1A11 K P W V V S Y IGkV3_11 R L LI A D F 1A11L0 R L L I A D F 1A11L1 R L W I A D F 1A11L2 R L L I A D Y1A11L3 R L W I A D Y 1A11L4 R P L I A D F 1A11L5 R P W I A D F 1A11L6 RP W V A D Y 1A11L7 K P W V A D Y 1A11L8 K P W V A S Y 1A11L9 K P W V V SY

2.0 Humanization of 6A3 2.1 6A3 Heavy chain Humanisation Strategy

Following a BLAST analysis of the human V gene germline databases, humangermline IGHV4_(—)61 which had 71% identity (including CDRs) with themouse 6A3 variable heavy chain sequence and human germline IGHV3_(—)33which had 51% identity (which has been previously shown to express wellwith IGKV1_(—)39) were selected as the preferred acceptor frameworks forhumanisation. The germline V region was combined in silico with asuitable FR4, in this case the JH6 minigene (Kabat Vol. II) based onsequence similarity. The first two residues of the JH6 minigene residuespreceding the WGQG motif fall within the CDR3 region which is replacedby the incoming CDR from the donor antibody. Ten humanised heavy chainvariants with framework IGHV4_(—)61 and twelve humanised heavy chainvariants with framework IGHV3_(—)33 were generated on the basis ofsequence comparison and possible impact on antibody function. ConstructH0 was a straight graft of mouse CDRs from 6A3 (using the Kabatdefinition) into the human acceptor framework selected above. H1 throughH5 constructs with framework IGHV4_(—)61 are based on H0, eachincorporates one additional framework mutation which was different ineach construct; positions 71, 27, 30, 67 and 48 respectively. H6 throughH9 constructs incorporate two, three, four or five of the above backmutations. H1 through H11 constructs with framework IGHV3-33 are basedon H0, each incorporates one additional framework mutation which wasdifferent in each construct; positions 27, 30, 28, 29, 67, 73, 78, 49,68, 24 and 48 respectively.

2.2 6A3 Heavy Chain Humanization Rationale for Framework IGHV4_(—)61

Kabat position 27 30 48 67 71 VH6A3 Y T M I R IGHV4_61 G S I V V 6A3H0 GS I V V 6A3H1 G S I V R 6A3H2 Y S I V V 6A3H3 G T I V V 6A3H4 G S I I V6A3H5 G S M V V 6A3H6 Y S I V R 6A3H7 Y T I V R 6A3H8 Y T I I R 6A3H9 YT M I R

2.3 6A3 Heavy Chain Humanization Rationale for Framework IGHV3_(—)33

Kabat position 27 28 29 30 48 49 67 68 73 78 VH6A3 Y S I T M G I S T FIGHV3_33 F T F S V A F T N L 6A3H0 F T F S V A F T N L 6A3H1 Y T F S V AF T N L 6A3H2 F T F T V A F T N L 6A3H3 F S F S V A F T N L 6A3H4 F T IS V A F T N L 6A3H5 F T F S V A I T N L 6A3H6 F T F S V A F T T L 6A3H7F T F S V A F T N F 6A3H8 F T F S V G F T N L 6A3H9 F T F S M A F S N L

2.4 6A3 Light Chain Humanisation Strategy

Following a BLAST analysis of the human V gene germline databases, humangermline IGKV1_(—)39 which had 72% identity (including CDRs) with themouse 6A3 variable light chain sequence was selected as the preferredacceptor framework for humanisation. The germline V region was combinedin silico with a suitable FR4, in this case the J-region kappa 2minigene (Kabat Vol. II) based on sequence similarity. The first tworesidues of the JK-2 minigene residues fall within the CDR3 region andare identical to the last two residues in the mouse 6A3 light chainCDR3. Five humanised light chain variants were generated on the basis ofsequence comparison and possible impact on antibody function. ConstructL0 was a straight graft of mouse CDRs from 6A3 (using the Kabatdefinition) into the human acceptor framework selected above. ConstructsL1, L2 are based on L0, each incorporates one additional frameworkmutation which were different in each construct; positions 45, 70respectively. Construct L3 incorporates both of the above backmutations.

6A3 Light Chain Humanization Rationale for Framework IGKV1_(—)39

Kabat position 45 70 Vk6A3 Q K IGkV1_39 K D 6A3L0 K D 6A3L1 Q D 6A3L2 KK 6A3L3 Q K

Five humanised light chain variants were generated on the basis ofsequence comparison and possible impact on antibody function. ConstructL0 was a straight graft of mouse CDRs from 6A3 (using the Kabatdefinition) into the human acceptor framework selected above. ConstructsL1, L2 are based on L0, each incorporates one additional frameworkmutation which were different in each construct; positions 45, 70respectively. Construct L3 incorporates both of the above backmutations; Construct L27 incorporates more mutations include T4L, A31Y,D70M, V85T, T94Y, Q100G, L104V (SEQ ID NO:138).

TABLE 3 6A3 variable light humanised variants Humanised Backmutations VLTemplate (Kabat#) L0 Straight graft of None 6A3VLCDRs onto IGKV1- 39 +JK-2 minigene L1 L0 K45Q L2 L0 D70K L3 L1 K45Q, D70K L27 L0 T4L, A31Y,D70M, V85T, T94Y, Q100G, L104V

2.5 Construction of Fc Disabled Variable Heavy Chain

Two amino acid substitutions, L237A and G239A were made to the 1A11 H3construct. These modifications render the molecule less able to recruitimmune effector cells or complement. The resulting VH construct isidentified as 1A11 H3-Fc, and has a sequence as shown in SEQ ID NO:118.The antibody comprising 1A11 VH3-Fc and the 1A11.L4 light chain (1A11H3L4Fc) was further analysed as described in Example 4 below.

2.6 Affinity Maturation of 1A11 H3L4

2.6.1 Construction of Recombinant Anti-IL7R 1A11 H3L4 CDRH3 Variants

A number of variants of the humanised anti-IL7R monoclonal antibody 1A11H3L4 were produced. These all differed by only one amino acidsubstitution in the CDRH3 region of the heavy chain of the antibodyhaving the heavy chain amino acid sequence set out in SEQ ID NO:114(H3), and the light chain amino acid sequence set out in SEQ ID NO:115(L4).

Humanised CDRH3 variants of 1A11 H3L4 within the pLEFD mammalianexpression vector were generated using site-directed mutagenesis.

2.6.2 Small scale antibody expression in HEK 293 6E cells

pLEFN and pLEFD plasmids encoding the light and heavy chains of 1A11H3L4 and CDRH3 variants respectively were transiently co-transfectedinto HEK 293 6E cells at 96-well scale (500 μl expression volume) using293fectin (Invitrogen, 12347019). Supernatants were harvested bycentrifugation for 10 minutes at 1500 rpm. The supernatants containingantibody were then filtered using a 0.45 μm filtration plate. Antibodieswere assessed directly from the tissue culture supernatant.

2.6.3 Proteon Analysis of Anti-IL7R 1A11 H3L4 CDRH3 Variants

The initial screen to determine the binding affinity of the CDRH3antibody variants (which were derived from small scale antibodyexpressions in HEK 293 6E cells and assessed directly from the tissueculture supernatant, as described in Example 2.6.2) was carried out onthe ProteOn XPR36 (Biorad). The method was as follows; Protein A wasimmobilised on a GLC chip by primary amine coupling, CDRH3 mutantantibodies were then captured on this surface and IL7R passed over at256, 64, 16, 4, 1 nM with a 0 nM injection (i.e. buffer alone) used todouble reference the binding curves. 50 mM NaOH was used to regeneratethe capture surface, removing the bound CDRH3 mutant antibodies readyfor another cycle of capture and analyte injection. The data was fittedto the 1:1 model using the analysis software inherent to the machine.Binding analysis for mutant antibodies was carried out directly fromtissue culture supernatants.

The screen identified several antibodies that appeared to have betterkinetic profiles than the parental molecule (1A11 H3L4). The dataobtained from this analysis is shown in 4.2.3, showing that severalCDRH3 mutations at the N98 and F100b residues appeared to improve thebinding affinity to IL7R. From this data set, six molecules wereselected for further analysis (Example 2.6.4).

2.6.4 Larger Scale Antibody Expression in HEK 293 6E Cells

The data highlighted that several CDRH3 mutations at the N98 and F100bresidues appeared to improve the binding affinity to IL7R (data shown in4.4.3). Therefore, purified antibody was produced for these six CDRH3variants. Constructs encoding the heavy and light chain of 1A11 H3L4CDRH3 variants were sub-cloned from the pLEFD and pLEFN plasmids intothe pTT vector for optimal large scale HEK 293 6E expression. Plasmidswere transiently co-transfected into 50-100 ml of HEK 293 6E (plasmiddetails summarised in Table 7) using 293fectin (Invitrogen, 12347019). Atryptone feed was added to the cell culture after 24 hours and the cellswere harvested after a further 72 hours. Antibody was then affinitypurified using immobilised Protein A columns and quantified by readingthe absorbance at 280 nm.

3.0 Construction of Humanised Vectors

The DNA sequences of the humanised variable regions were sequenceoptimised using the LETO 1.0 software (Entelechon GmbH) and synthesisedde novo by build up of overlapping oligonucleotide and PCRamplification. Primers included restriction sites for cloning intomammalian expression vectors and human immunoglobulin signal sequencesfor secretion. The humanised variable heavy regions were cloned intomammalian expression vectors containing the human gamma 1 constantregion using Age1/Kas1. In parallel, the humanised variable lightregions were cloned into mammalian expression vectors containing thehuman kappa constant region using HindIII and BsiWI.

4.0 Characterisation of Humanised Antibodies 4.1 Determination ofBinding Kinetics of 1A11 and 6A3 Constructs: BIACORE™ 3000

The binding kinetics of the anti-CD127 antibodies for human CD127 ECDwas assessed using a BIACORE™ 3000 device (GE Healthcare). Humanised 6A3or 1A11 constructs were captured on a CM5 biosensor chip which wasalready immobilized BIAcore (GE Healthcare cat# BR-1008-39) anti-humanIgG (Fc specific) monoclonal antibody using supplied coupling buffer. Arange of human CD127 ECD concentrations (512, 256, 128, 64, 32, 16 nM)were injected for 240 s at a flow rate of 30 ul/min.

-   -   1) Capture MAb of interest    -   2) Association of Analyte to captured MAb    -   3) Dissociation of Analyte (buffer)    -   4) Regenerate with BIAcore optimized buffer. Removes all but        covalently coupled anti-H Ab. BIAcore Kinetic run Cycles:        buffer, 512, 256, 128, 64, 32, 16 nM IL7R ECD; buffer cycle used        for double referencing.

The antibody surfaces were regenerated with 3 M MgCl₂. Kinetics weredetermined by global fitting of data to the 1:1 Langmuir model usingBIAEVALUATION software. Results are shown in Example 4.2.1 (1A11) and4.2.2 (6A3).

4.1.1 Determination of Binding Kinetics of Selected 1A11 H3L4 CDRH3Variants

BIACORE™ analysis was used to determine the binding affinity of thepurified CDRH3 mutant antibodies (which were derived from larger scaleantibody expressions in HEK 293 6E cells, as described in Example2.6.4).

4.1.1.1 Method 1: BIACORE™ T100

An anti-human IgG (GE Healthcare/BIAcore™ BR-1008-39) was immobilised ona CM3 chip by primary amine coupling to a level of ˜1300 resonance units(RU's), CDRH3 mutant antibodies were then captured on this, all theantibodies were captured to a similar level (44-56 RU's) and IL-7Rpassed over at 256, 64, 16, 4, 1 nM with a 0 nM injection (i.e. bufferalone) used to double reference the binding curves, regeneration of thissurface was achieved using 3M MgCl₂. The binding data was fitted to the1:1 model inherent to the BIAcore™ T100 analysis software. The run wascarried out using HBS-EP as running buffer and carried out at 25° C. onthe BIAcore™ T100. Results are shown in 4.2.4.

4.1.1.2: Method 2: BIACORE™ 3000

An anti-human IgG (GE Healthcare/BIACORE™ BR-1008-39) was immobilised ona CM5 chip by primary amine coupling to a level of ˜5400 resonance units(RU's), CDRH3 mutant antibodies were then captured on this surface, allthe antibodies were captured to a similar level (175-205 RU's) and IL7Rpassed over at 64, 16, 4, 1 nM with a 0 nM injection (i.e. buffer alone)used to double reference the binding curves, regeneration of thissurface was achieved using 3M MgCl₂. The binding data was fitted to the1:1 model inherent to the BIACORE™ 3000 analysis software. The run wascarried out using HBS-EP as running buffer and carried out at 25° C. onthe BIACORE™ 3000. Results are shown in 4.2.5.

4.1.2 Determination of species cross-reactivity of 1A11 H3L4 inCynomolgus and Marmoset

The binding kinetics of 1A11H3L4 for Cynomolgus and Marmoset CD127 ECDwas assessed using a BIACORE 3000. 1A11H3L4 was captured on a CM5biosensor chip which was already immobilized BIACORE™ (GE Healthcarecat# BR-1008-39) anti-human IgG (Fc specific) monoclonal antibody. Theantibody surfaces were regenerated with 3 M MgCl₂. Kinetics wasdetermined by global fitting of data to the 1:1 Langmuir model usingsoftware. Results are shown in 4.3.

4.1.3 IL7 Receptor Inhibition Assay

BIACORE™ analysis was also used to demonstrate that the purified CDRH3mutant antibodies (which were derived from larger scale antibodyexpressions in HEK 293 6E cells, as described in Example 2.6.4) wereable to inhibit the interaction between 17 and IL7R.

IL7 (R&D Systems) was immobilised on a CM5 chip by primary aminecoupling; the surface was conditioned with 10 mM glycine, pH3.0 toprovide a stable surface for the neutralisation assay. IL7R at 64 nM wasincubated with the test antibodies at concentrations of 256 nM, 128 nM,64 nM, 16 nM, 8 nM, 4 nM, 2 nM and 1 nM in run 1 and of 256 nM, 128 nM,64 nM, 16 nM, 8 nM, 4 nM, 2 nM, 1 nM, 0.5 nM and 0.25 nM in run 2.Samples were then incubated at room temperature for 3 hrs before beingrun over the IL7/CM5 chip, 10 mM glycine, pH3.0 was used to regeneratethe surface for the next interaction. IC₅₀ values were calculated usingRobosage, whereby the binding signals were converted into percentagevalues based around the maximum signal achieved using IL7R at 64 nM with0 nM antibody. Results are shown in 4.7.

4.2 Binding Kinetics Results

4.2.1 1A11

TABLE 4 Binding kinetics for 1A11 constructs Sample ka (1/Ms) kd (1/s)KD (M) 1A11 Chimera 9.26e4 2.98e−4 3.22e−9 1A11 H0L0 No binding seen — —1A11 H1L1 No binding seen — — 1A11 H6L6 No binding seen — — 1A11 H7L5 Noexpression seen — — 1A11 H3L4 1.77e5 4.64e−4 2.62e−9 1A11 H3L5 2.94e46.07e−3 2.07e−7 1A11 H3L9 4.32e4 1.84e−3 4.25e−8 1A11 H3L6 1.82e42.82e−3 1.55e−7 1A11 H4L4 No binding seen — — 1A11 H6L4 No binding seen— — 1A11 H7L4 No expression seen — — Biotin-labelled 1.69e5 4.52e−42.67e−9 1A11 H3L4 1A11 H3L4Fc 1.8e5  6.62e−4 3.68e−94.2.2 6A3

TABLE 5 Binding kinetics for 6A3 constructs Sample ka (1/Ms) kd (1/s) KD(M) H4-61_6L27 1.6e4 2.99e−4 19 H4-61_7L27 3.77e4 1.04e−3 28 H4-61_8L272.43e4 3.7e−4 15 H4-61_9L27 6.99e4 1.22e−3 184.2.3 Selection of Various Anti-IL7R 1A11 H3L4 CDRH3 Variants of 1A11H3L4 by BIACORE™ Analysis

TABLE 6 Proteon analysis of anti-IL-7R 1A11 H3L4 CDRH3 variants (KD, innM)

Representative KD value for 1A11 H3L4 = 0.116 nM

TABLE 7 Selected CDRH3 variant mAbs constructed and expressed DNAProtein Antibody Alternative Batch sequence sequence ID names No.Molecule description ID No. ID No. BPC4398 1A11 H3L4 N98D HEK1023 Hchain: Anti-human IL7R 120 121 (CDRH3 variant) 1A11 VH3 N98D L chain:Anti-human IL7R 108 22 1A11 VL4 BPC4399 1A11 H3L4 N98E HEK1024 H chain:Anti-human IL7R 122 123 (CDRH3 variant) 1A11 VH3 N98E L chain:Anti-human IL7R 108 22 1A11 VL4 BPC4400 1A11 H3L4 HEK1025 H chain:Anti-human IL7R 124 125 F100bE (CDRH3 1A11 VH3 F100bE variant) L chain:Anti-human IL7R 108 22 1A11 VL4 BPC4401 1A11 H3L4 HEK1026 H chain:Anti-human IL7R 126 127 F100bH (CDRH3 1A11 VH3 F100bH variant) L chain:Anti-human IL7R 108 22 1A11 VL4 BPC4402 1A11 H3L4 HEK1027 H chain:Anti-human IL7R 128 129 F100bI (CDRH3 1A11 VH3 F100bI variant) L chain:Anti-human IL7R 108 22 1A11 VL4 BPC4403 1A11 H3L4 HEK1028 H chain:Anti-human IL7R 130 131 F100bV (CDRH3 1A11 VH3 F100bV variant) L chain:Anti-human IL7R 108 22 1A11 VL4 BPC1142 1A11 H3L4 HEK1029 H chain:Anti-human IL7R 13 32 1A11 VH3 GRITS37988 L chain: Anti-human IL7R 10822 1A11 VL44.2.4 BIACORE™ T100 analysis of selected 1A11 H3L4 CDRH3 variants

Table 8 shows the data obtained from the 4.1.1.1 study, which shows thatall the CDRH3 mutations appeared to have better affinities than theparental molecules with the best construct appearing to be BPC4398(Anti-IL7R 1A11 H3L4 N98D).

TABLE 8 Molecule Molecule ka Kd KD identifier/number description (M/s)(1/s) (nM) BPC1142 Anti-IL7R 1A11 1.08E+06 7.06E−05 0.065 (GRITS37988)H3L4 Replicate 1 BPC1142 Anti-IL7R 1A11 1.12E+06 5.86E−05 0.052(GRITS37988) H3L4 Replicate 2 BPC4398 Anti-IL7R 1A11 1.71E+06 3.70E−050.022 H3L4 N98D (CDRH3 variant) BPC4399 Anti-IL7R 1A11 1.45E+06 4.08E−050.028 H3L4 N98E (CDRH3 variant) BPC4400 Anti-IL7R 1A11 9.24E+05 2.68E−050.029 H3L4 F100bE (CDRH3 variant) BPC4401 Anti-IL7R 1A11 9.10E+053.06E−05 0.034 H3L4 F100bH (CDRH3 variant) BPC4402 Anti-IL7R 1A118.26E+05 3.32E−05 0.040 H3L4 F100bI (CDRH3 variant) BPC4403 Anti-IL7R1A11 8.47E+05 3.30E−05 0.039 H3L4 F100bV (CDRH3 variant) BPC1142Anti-IL7R 1A11 1.16E+06 6.48E−05 0.056 (HEK1029) H3L4 The parentalmolecule (BPC1142-anti-IL7R 1A11 H3L4) was run multiple times within theexperiment using CHO expressed material (GRITS37988)) and HEK expressedmaterial (HEK1029), no significant difference was seen betweenaffinities for the different expression systems for the parentalmolecule.4.2.5 BIACORE™ 3000 analysis of selected 1A11 H3L4 CDHR3 variants

Table 9 shows the data obtained from the 4.1.1.2 study and shows thatall the CDRH3 mutations appeared to have better affinities than theparental molecules with the best constructs appearing to be BPC4398(1A11 H3L4 N98D) and BPC4399 (1A11 H3L4 N98E).

TABLE 9 Molecule Molecule ka kd KD identifier/number description (M/s)(1/s) (nM) BPC1142 Anti-IL7R 1A11 3.60E+05 2.70E−04 0.751 (GRITS37988)H3L4 Replicate 1 BPC1142 Anti-IL7R 1A11 3.62E+05 2.36E−04 0.651(GRITS37988) H3L4 Replicate 2 BPC4398 Anti-IL7R 1A11 5.44E+05 2.09E−040.385 H3L4 N98D (CDRH3 variant) BPC4399 Anti-IL7R 1A11 5.97E+05 2.33E−040.39 H3L4 N98E (CDRH3 variant) BPC4400 Anti-IL7R 1A11 3.51E+05 1.91E−040.546 H3L4 F100bE (CDRH3 variant) BPC4401 Anti-IL7R 1A11 3.37E+051.96E−04 0.582 H3L4 F100bH (CDRH3 variant) BPC4402 Anti-IL7R 1A113.00E+05 2.00E−04 0.668 H3L4 F100bI (CDRH3 variant) BPC4403 Anti-IL7R1A11 2.98E+05 1.89E−04 0.636 H3L4 F100bV (CDRH3 variant) BPC1142Anti-IL7R 1A11 3.49E+05 2.29E−04 0.656 (HEK1029) H3L4 The parentalmolecule (BPC1142-anti-IL7R 1A11 H3L4) was run multiple times within theexperiment using CHO expressed material (GRITS37988) and HEK expressedmaterial (HEK1029), no significant difference was seen betweenaffinities for the different expression systems for the parentalmolecule.

Differences were seen in the overall affinities calculated between thetwo methods. This is likely to be due to the fact that IL7R is ahomodimer and therefore the amount of avidity and cross linking of theantibodies with the antigen may increase or decrease dependent on thedifferent densities of IL7R immobilised by the different capturesurfaces used in the two assays. Despite the different affinities seenbetween the two runs the ranking in the two experiments shows thatBPC4398 (1A11 H3L4 N98D) has a better affinity than the parentalmolecule 1A11 H3L4.

4.3 Species Cross-Reactivity

1A11 H3L4 (wild type) was observed to cross react with marmoset andcynomolgus IL-7R tested at a comparable level by BIACORE™ system (Table10).

TABLE 10 1A11 H3L4 with Human IL7R, Mouse IL7R and Cynomolgus IL7RComparison Sample ka (1/Ms) kd (1/s) KD (M) 1A11 H3L4 with 1.77e54.64e−4 2.62e−9 Human IL7R 1A11 H3L4 with 2.58e4 2.34e−4 9.06e−9Cynomolgus IL7R 1A11 H3L4 with 4.93e4 2.99e−4 6.05e−9 Marmoset IL7R

4.4 Epitope Binding by X-Ray Crystallography

Using a 1A11 H3L4 Fab, X-ray crystallography coupled with in silicomodelling was used to predict binding interfaces for the mAbs to helpprovide mechanistic insight into the functional neutralization observed,and to make rational choices for antibody maturation. A high resolution(2.08 A) structure of 1A11H3L4 Fab/human IL7 receptor complex wasestablished. Human IL7 Receptor extracellular domain and 1A11H3L4 wereexpressed in CHO lec cells and purified by affinity chromatography andsize exclusion chromatography. The Fab fragment of 1A11H3L4 wasgenerated by papain cleavage. Fab1A11H3L4/IL7R ECD complex was generatedby mixing 1:1.2 molar ratio of Fab1A11H3L4 with IL7Receptor ECD.Proteins were concentrated and crystallized using the hanging drop vapordiffusion method. X-ray diffraction data were collected at the AdvancedPhoton Source in the Argonne National Laboratory. Diffraction data wereindexed and scaled using the HKL2000 software. The structure wasdetermined by molecular replacement in the program X-PLOR. The initialmolecular replacement solution was subject to multiple rounds ofmolecular dynamics refinement in CNX and rebuilding with the programWinCoot.

Based on the high resolution 2.08 A crystal structure, it is predictedthat 1A11H3L4 binds IL7 Receptor at 4 of the IL-7R extracellular loops,thereby blocking IL7-ligand binding:

-   -   Loop 2: 55Gly 56Ala 57Leu 58Val 59Glu 60Val 61Lys    -   Loop 3: 80Leu 81Leu 82Ile 83Gly 84Lys 100Lys    -   Loop 4: 138Lys 139Tyr 142Val    -   Loop 5: 192Tyr 193Phe

These findings are consistent with the observed competition for bindingto hIL7 observed between 1A11 and 6A3.

4.5 Analysis of Effector Functions

4.5.1 1A11 H3L4 Lacks Complement-Mediate Cytotoxicity

A total of six separate experiments showed that 1A11 H3L4 (wild type)had no measurable complement-mediated cytotoxicity. These experimentswere performed with a hIL-7r BacMam transduced HEK 293 MSR II cell lineused as the target. These cells were transduced (moi 75) for ˜21 hoursat 37° C., 5% CO₂ in T175 culture flasks. The adherent cells were thenremoved from the flasks using TrypLE and washed several times beforeplating at 1×10⁵ cells/50 ul/well into a 96-well plate. 25 μl ofantibody was added for 30 minutes at 37° C., 5% CO₂. Following thisincubation, 20 μl of rabbit complement was added and the plate and thenreturned to the incubator for 2 hours. An assessment of cell viabilitywas carried out by adding 100 μl of CellTiter-Glo to each well withgentle mixing using a multichannel pipet. The plate was then read forluminescence signal on a Victor V plate reader (viable cells haveincreased signal). An example of one of those experiments is shown inFIG. 1.

The positive control antibody (Grits 32092) used in the above experimentwas specific towards a cell-surface receptor for HER3 that wasco-expressed on the same target cell which expressed the hIL-7Rα. Thiscontrol antibody was used at the same concentration as 1A11 H3L4 (10μg/ml), and was combined with the same two sources of rabbit complement(Calbiochem and Invitrogen). These results showed that both the targetcells and complement which were used in the assay were able to inducecomplement dependent cytotoxicity.

4.5.2 1A11 H3L4Fc has Reduced Antibody Dependent Cell-MediatedCytotoxicity (ADCC)

Purified peripheral blood mononuclear cells from seven human donors wereprofiled as effector cells in an ADCC assay. These experiments wereperformed with a hIL-7r BacMam transduced HEK 293 MSR II cell line usedas the target cell. These cells were transduced (moi 75) for ˜21 hoursat 37° C., 5% CO₂ in T175 culture flasks. These adherent cells were thenremoved from the flasks using Tryple and washed several times before“loading” with europium. These loaded cells were combined into a 96-wellplate (2×10⁴ cells/25 ul/well) which contained anti-IL-7R antibody for30 minutes at 37° C., 5% CO₂. After incubation, effector cells wereadded at ratios of 200, 100, 50 and 25:1 (100 μl/well) and returned to37° C., 5% CO₂ for 2 hours. Following this incubation, 25 μl ofsupernatant was removed and added to a 96-well plate containing 100μl/well of Delfia enhancement solution. The plate was then incubated ona room temperature plate shaker for 5 minutes and then read in a VictorV plate reader. Any europium released by lysed cells into thesurrounding supernatant (cell cytotoxicity) was measured as fluorescentunits.

These assays compared the ability of “wild-type” 1A11 H3L4 and theFc-disabled molecule 1A11 H3L4Fc to bind human effector cells via theirFc receptors, and kill IL-7 receptor positive target cells. The overallresults from these experiments showed that the Fc-disabled 1A11 H3L4Fcwas at least 2-fold less potent in initiating antibody dependentcell-mediated cytotoxicity than “wild-type” 1A11 H3L4. These resultsalso showed that in six out of seven donors the disabled antibody wascapable of inducing some level of ADCC activity (one donor showed littleactivity with both wild-type and disabled 1A11 H3L4). The results areshown in FIG. 2.

4.5.3 Fc Receptor Binding

1A11 H3L4 and 1A11 H3L4Fc were assessed for their ability to bind tomultiple Fc effector receptors (Fc Gamma I, IIa and IIIa), and FcRn ofnumerous species and compared to control wild-type and Fc disabledantibodies. The work was carried out on the ProteOn XPR36 surfaceplasmon resonance machine (BioRad). The antibodies to be tested werecoupled to a GLM biosensor chip by primary amine coupling. The variousFcγ receptors were used as analytes at 2048 nM, 512 nM, 128 nM, 32 nMand 8 nM using HBS-EP (pH7.4) as running buffer. For FcRn receptorbinding, human, cyno, mouse and rat FcRn were used as analyte at 2048nM, 512 nM, 128 nM, 32 nM and 8 nM, with the run being carried out atpH6.0 and pH7.4. All binding sensograms were double referenced with a 0nM injection (i.e. buffer alone). The data was fitted to the Equilibriummodel inherent to the ProteOn analysis software.

Table 11 shows the affinities generated for the antibody binding to thevarious Fc receptors assessed in this study, and shows that 1A11 H3L4and 1A11 H3L4Fc behaved in a comparable manner to their control antibodycounterparts. The disabled Fc antibodies (1A11 H3L4Fc) showed either nobinding or a much reduced binding for Fcγ receptors and hence accurateanalysis could not be carried out. The data for FcRn binding showed thatthe Fc disabled and Fc wild type had similar affinities for all speciestested, the data in the table is for the pH6.0 assay, binding was eitherabsent or much reduced at pH7.4 as expected.

TABLE 11 Binding affinities of 1A11 H3L4 (Fc disabled and wt Fc) to FcReceptors (nM) Fcγ2a Fcγ2a Fcγ3a Fcγ Human Cyno Mouse rat Constructs(Arg) (His) (Phe) (Val) Fcγ1 FcRn FcRn FcRn FcRn Control 1290 1500 1840442 14.9 95 156 160 112 Ab (Fc WT) Control Much no no no Much 154 195171 118 Ab (Fc Reduced binding binding binding Reduced disabled) BindingBinding 1A11 1250 1040  990 319 21.4 183 210 192 145 H3L4 1A11 Much nono no Much 158 248 207 163 H3L4Fc Reduced binding binding bindingReduced Binding Binding

4.6 In Vitro Potency Assays

4.6.1 Inhibition of IL-7 Stimulated STAT5 Phosphorylation by 1A11 and1A11 H3L4

For screening functional antibody to IL-7Rα, hybridoma culture medium,positive control antibody or testing supernatant samples were incubatedwith PBMC cells for 30 mins before stimulating with IL-7. The untreatedcells were analyzed as the background signal, while IL-7 treated cellswere set as negative control. After 30 mins incubation with the controlsor testing samples, the cells were stimulated with IL-7 for 15 mins at37° C. Cells then were fixed 1.6% of paraformaldehyde/PBS for 10 min at37° C. and were permeabilized in 100% methanol for 20-30 mins. Cellsthen were washed twice in stain buffer (1% BSA in PBS) and stained withAlexa-647 labelled anti-pStat5 antibody (BD Biosciences Inc #612599) for1 hr. Samples were analyzed on BD LSR II FACS instrument.

The parental 1A11 monoclonal antibody blocks IL-7 induction of STAT5phosphorylation in human PBMC with an IC50 of 0.088 ug/ml (data notshown). 1A11 H3L4 was tested in the same assay using PBMCs from twodonors at two human IL-7 concentrations (0.1 ng/ml and 1 ng/ml). 1A11H3L4 demonstrated a very similar IC50 (average=0.087 ug/ml) compared to1A11 indicating that the humanization process did not affect the abilityof the antibody to inhibit IL-7-induced pSTAT5 (FIGS. 3A and 3B).

4.6.2 Inhibition of IL-7-Induced IL-17 Production by 1A11 H3L4

1A11 H3L4 was assayed according to the following protocol, to determineits ability to inhibit Th17 expansion. CD4+ cells were isolatedaccording to the manual (#130-091-155, Miltenyi). Approximately 1×10⁶/mlof the CD4+ cells in 100 μl were mixed with equal volume of a 2×concentration of Th17 differentiation medium (2 μg/ml anti-CD28+10 μg/mlanti-IFN-γ+10 μg/ml anti-IL-4+12.5 ng/ml IL-1β+20 ng/ml IL-23+50 ng/mlIL-6) and cultured in 37° C. with 5% CO₂ for 5 days. Treatment by thevarious cytokines and growth factors in the Th17 medium preferentiallydifferentiated the CD4+ cells into Th17 cells. CCR6+ cells from thedifferentiated cultured cells at day 5 were sorted using BD FACS SORPAria II. The CCR6+ cells were then adjusted to 2×10⁶/ml for the IL-17production assay.

To measure IL-17 and IFN-γ level, 100 μl of CCR6+ cells werepre-incubated with testing antibody for 1 h at 37° C., and then mixedwith 100 μl of 10 ng/ml IL-7. The cells were cultured for 24-40 hours in37° C. with supplement of 5% CO₂. IFN-γ and IL-17 levels in 100 μl ofculture supernatant were measured by FlowCytomix (Bender MedSystems) at24 h and 40 h, respectively.

1A11 H3L4 was tested in the Th17 expansion assay in a total of fourhuman CD4+ cell samples (FIGS. 4A-D). The humanized antibodydemonstrated significant inhibition of IL-17 production in two samplesand trends of inhibition in the other two samples. Given thedonor-to-donor variation in this assay, we conclude that 1A11 H3L4 isable to block IL-7-mediated Th17 cell expansion.

4.6.3 Effects on TSLP Signalling

The IL-7Rα subunit is shared by both the IL-7R and the TSLP receptorcomplex (TSLPR). The effect of 1A11 H3L4 on TSLP signaling was tested inan in vitro assay based on TSLP-induction of TARC production by humanblood monocytes. A commercial anti IL-7Rα antibody, R34.34, was used asa positive control for blocking TSLP-induction of TARC. Furthermore anFc-disabled humanized IgG1 (HuIgG1; GRITS39633) was also used as anegative control. Monocytes from 5 donors were used, with TSLP used at 1ng/ml, 1A11 H3L4 and HuIgG1 used at doses of 0.001-30 μg/ml, and R34.34used at 0.4, 2, and 10 μg/ml. Cell survival was also assessed by cellcounting.

1A11 H3L4 did not affect TSLP-induction of TARC as shown in FIGS. 5A-E,whilst for the same 5 donors, R34.34 substantially inhibited TARCproduction. The humanized negative control antibody had no effect onTARC production. This data set shows that 1A11 H3L4 does not neutralizeTSLP signaling in human monocytes. Therefore it is anticipated that 1A11H3L4 is specific for neutralization of IL-7 signaling through IL-7R anddoes not impact upon TSLP signaling through TSLPR.

4.7 IL7 Receptor Inhibition Assay

Table 12A shows the IC₅₀ values obtained in run 1 and shows that all ofthe constructs had better IC₅₀ values than the best value obtained forthe parental 1A11 H3L4 molecule and that the top two molecules wereBPC4401 (Anti-IL7R 1A11 H3L4 F100bH) and BPC4398 (1A11 VH3 N98D L4).Table 12B shows the IC₅₀ values obtained in run 2, and also shows thatboth the constructs, BPC4401 (Anti-IL7R 1A11 H3L4 F100bH) and BPC4398(1A11 VH3 N98D L4) had better IC₅₀ values than the best value obtainedfor the parental 1A11 H3L4 molecule. Run's 1 and 2 were carried out onseparate IL7R/CM5 surfaces.

TABLE 12A IC50s Receptor inhibition assay (Run1) Moleculeidentifier/number Molecule description IC50 (nM) BPC1142(GRITS37988)Anti-IL7R 1A11 H3L4 19.42 Replicate 1 BPC4398 Anti-IL7R 1A11 H3L4 N98D14.94 (CDRH3 variant) BPC4399 Anti-IL7R 1A11 H3L4 N98E 15.8 (CDRH3variant) BPC4400 Anti-IL7R 1A11 H3L4 F100bE 15.33 (CDRH3 variant)BPC4401 Anti-IL7R 1A11 H3L4 F100bH 14.75 (CDRH3 variant) BPC4402Anti-IL7R 1A11 H3L4 F100bI 15.23 (CDRH3 variant) BPC4403 Anti-IL7R 1A11H3L4 F100bV 15.54 (CDRH3 variant) BPC1142 (HEK1029) Anti-IL7R 1A11 H3L416.21 BPC1142 (GRITS37988) Anti-IL7R 1A11 H3L4 17.23 Replicate 2 Theparental molecule (BPC1142-anti-IL7R 1A11 H3L4) was run multiple timeswithin the experiment using CHO expressed material (GRITS37988) and HEKexpressed material (HEK1029).

TABLE 12B IC50s Receptor inhibition assay (Run 2) Moleculeidentifier/number Molecule description IC₅₀ (nM) BPC1142(GRITS37988)1A11 H3L4 16.44 Replicate 1 BPC4398 Replicate1 1A11 VH3 N98D L4 14.86BPC4401 Replicate1 1A11 VH3 F100bH L4 14.92 BPC1142(GRITS37988) 1A11H3L4 16.57 Replicate 2 BPC1142(GRITS37988) 1A11 H3L4 16.79 Replicate 3BPC4398Replicate 2 1A11 VH3 N98D L4 14.17 BPC4401 (Replicate 2 1A11 VH3F100bH L4 15.15 BPC1142(GRITS37988) 1A11 H3L4 17.35 Replicate 4 Theparental molecule (BPC1142-anti-IL7R 1A11 H3L4) was run multiple timeswithin the experiment using CHO expressed material (GRITS37988),replicate runs of BPC4398 (1A11 VH3 N98D L4) and BPC4401 (1A11 VH3F100bH L4).

4.8 IL-7R Polymorph Binding Assay

IL-7R exists as two polymorphic forms, variant 1: Thr66-Ile128, variant2: Ile66-Thr128. Binding of 1A11H3L4 to both polymorphic forms wasassayed. An anti-human IgG (GE Healthcare/BIACORE™ BR-1008-39) wasimmobilised on a CM5 chip by primary amine coupling to a level of ˜9000resonance units (RU's), 1A11H3L4 was then captured on this surface, andIL7R passed over at 512 n, 256 n, 128 n, 64 nnM, 32 nM and 16 nM with a0 nM injection (i.e. buffer alone) used to double reference the bindingcurves, regeneration of this surface was achieved using 3M MgCl2. Thebinding data was fitted to the 1:1 model inherent to the BIACORE™ 3000analysis software. The run was carried out using HBS-EP as runningbuffer and carried out at 25° C. on the ™3000. Table 13 shows the dataobtained and showed that 1A11 H3L4 had the same or similar bindingaffinity to both polymorphic variants (i.e. it binds to bothpolymorphs).

TABLE 13 IL-7R polymorph binding 1A11H3L4 with hIL7R variant1: Kon (Ka)Koff (Kd) KD Thr66-Ile128 1.52e5 4.15e−4 2.73e−9 1A11H3L4 with hIL7Rvariant2: Kon (Ka) Koff (Kd) KD ILE66-Thr128 1.46e5 4.56e−4 3.1e−9

Within this specification the invention has been described, withreference to embodiments, in a way which enables a clear and concisespecification to be written. It is intended and should be appreciatedthat embodiments may be variously combined or separated without partingfrom the invention.

What is claimed is:
 1. An isolated antigen-binding protein comprising aheavy chain variable domain comprising the amino acid sequence set forthin SEQ ID NO:13, and a light chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO:22.
 2. The antigen-binding proteinof claim 1, which comprises a heavy chain amino acid sequence selectedfrom the group consisting of: SEQ ID NO:114 and SEQ ID NO:118.
 3. Theantigen-binding protein of claim 1, which comprises a light chain havingthe amino acid sequence set forth in SEQ ID NO:115.
 4. A pharmaceuticalcomposition comprising an antigen-binding protein according to claim 1,and a pharmaceutically acceptable carrier or excipient.
 5. A method oftreating a subject having multiple sclerosis, comprising the step ofadministering to the subject a therapeutically effective amount of anantigen-binding protein according to claim 1.